HEPATITIS D VIRUS (HDV) iRNA COMPOSITIONS AND METHODS OF USE THEREOF

ABSTRACT

The present invention relates to RNAi agents, e.g., double-stranded RNAi agents, targeting the hepatitis D virus (HDV) genome, and methods of using such RNAi agents to inhibit expression of one or more HBV genes and methods of treating subjects having an HDV infection and/or HDV-associated disorder.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/591,558, filed on May 10, 2017, which is a 35 § U.S.C. 111(a)continuation application which claims the benefit of priority toPCT/US2015/059958, filed on Nov. 10, 2015, which claims priority to U.S.Provisional Application, 62/077,672, filed on Nov. 10, 2014. The entirecontents of each of the foregoing patent applications are incorporatedherein by reference.

This application also claims priority to U.S. Provisional Application,62/077,799, filed on Nov. 10, 2014 and U.S. Provisional Application,62/137,464, filed on Mar. 24, 2015. The entire contents of each of theforegoing patent applications are incorporated herein by reference.

This application is related to International Patent Application,PCT/US2015/059916, filed on Nov. 10, 2015 and U.S. patent applicationSer. No. 15/591,532, filed on May 10, 2017. The entire contents of eachof the foregoing patent applications are incorporated herein byreference.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Feb. 25, 2020, isnamed 121301_02803_SL.txt and is 774,863 bytes in size.

BACKGROUND OF THE INVENTION

Hepatitis D virus or hepatitis delta virus (HDV) is a human pathogen.Indeed HDV infection is highly endemic to several African countries, theAmazonian region, and the Middle East, while its prevalence is low inindustrialized countries, except in the Mediterranean. However, thevirus is defective and depends on obligatory helper functions providedby the hepatitis B virus (HBV) for transmission; indeed, HDV requires asimultaneous infection with HBV (co-infection) or superimposition on apre-existing HBV infection (superinfection) to become infectious andthrive. In particular, HDV requires the HBV viral envelope containingthe surface antigen of hepatitis B.

HDV is unique in human virology; it has a circular RNA genome of about1,700 bases (see, e.g., Genbank Accession No. M21012.1 (GI:329989)) andis therefore the smallest infectious agent in man, and is similar toviroids and satellite RNAs of plants. HDV replicates by a rolling circlemechanism unknown to animal cells, possesses a self-cleaving ribozymeand is transcribed by host RNA polymerases that normally accept only DNAtemplates (Ciancio and Rizzetto, Nat. Rev. 11:68-71, 2014).

HDV is a circular, single stranded RNA virus that ranges from 1,672(strain dFr45, Genbank accession number AX741144) to 1,697 nucleotides(dFr47, GenBank accession number AX741149). A unique open reading frameencodes the small and large hepatitis delta (sHD and 1HD, respectively)antigens by way of an editing step in the hepatocyte nucleus.

The genetic diversity of HDV is related to the geographic origin of theisolates and there are at least eight genotypes that are referred to asHDV-1 through HDV-8. Apart from HDV-1, which is ubiquitous, HDV-2(previously labeled HDV-IIa) is found in Japan, Taiwan, and Yakoutia,Russia; HDV-4 (previously labeled HDV-IIb) in Taiwan and Japan; HDV-3which causes epidemics of severe and fulminant hepatitis in theAmazonian region (9); and HDV-5, HDV-6, HDV-7, and HDV-8 in Africa(LeGal et al., Emerg. Infect. Dis. 12:1447-1450, 2006).

Worldwide more than 400 million people are chronically infected withHBV. Furthermore, at the end of the 1980s, at least 5% of hepatitis Bsurface antigen (HBsAg) carriers throughout the globe (˜15 millionindividuals) were estimated to also be infected with HDV. Subjectsinfected with HBV are at increased risk of developing serious liverdisease, such as chronic hepatitis, cirrhosis, liver failure andhepatocellular carcinoma (HCC) resulting in an estimated 600,000 deathseach year, HDV infection in such subjects (either co-infection orsuperinfection) can lead to severe acute and chronic forms of liverdisease in association with HBV. In fact, both superinfection andcoinfection with HDV results in more severe complications compared toinfection with HBV alone. These complications include resistance totreatment with standard therapies and a greater likelihood ofexperiencing liver failure in acute infections and a rapid progressionto liver cirrhosis, with an increased chance of developing liver cancerin chronic infections. In combination with hepatitis B virus, hepatitisD has the highest fatality rate of all the hepatitis infections, at 20%.

Accordingly, there is a need in the art for alternative therapies andcombination therapies for subjects infected with HDV and/or having anHDV-associated disease.

SUMMARY OF THE INVENTION

The present invention provides iRNA compositions which effect theRNA-induced silencing complex (RISC)-mediated cleavage of RNAtranscripts of a Hepatitis D virus (HDV) gene. The HDV gene may bewithin a cell, e.g., a cell within a subject, such as a human.

The present invention also provides methods and therapies for treating asubject having a disorder that would benefit from inhibiting or reducingthe expression of an HDV gene, e.g., an HDV infection and/or anHDV-associated disease, such as hepatitis B virus infection, chronichepatitis B infection (CHB), chronic Hepatitis D infection (CHD),cirrhosis, liver failure, and hepatocellular carcinoma (HCC), using iRNAcompositions which effect the RNA-induced silencing complex(RISC)-mediated cleavage of RNA transcripts of an HDV gene forinhibiting the expression of an HDV gene. As HDV depends on obligatoryhelper functions provided by HBV for transmission, the methods fortreatment of HDV can comprise agents and therapies useful for treatingan HBV infection and/or an HBV-associated disorder, such as hepatitis Dvirus infection, chronic hepatitis D infection (CHD), chronic HepatitisB infection (CHB), cirrhosis, liver failure, and hepatocellularcarcinoma (HCC).

In certain embodiments, the RNAi agents of the invention have beendesigned to target regions in the HDV genomes that are conserved acrossat least two, preferably 3, or more of the 8 serotypes of HDV (HDV-1,HDV-2, HDV-3, HDV-4, HDV-5, HDV-6, HDV-7, and HDV-8).

Accordingly, in one aspect, the present invention provides doublestranded RNAi agents for inhibiting expression of hepatitis D virus(HDV) in a cell. The double stranded RNAi agents include a sense strandand an antisense strand forming a double-stranded region, wherein thesense strand comprises at least 15 contiguous nucleotides differing byno more than 3 nucleotides from the nucleotide sequence of SEQ ID NO:29,and the antisense strand comprises at least 15 contiguous nucleotidesdiffering by no more than 3 nucleotides from the nucleotide sequence ofSEQ ID NO:30, wherein substantially all of the nucleotides of the sensestrand and substantially all of the nucleotides of the antisense strandare modified nucleotides, wherein the sense strand is conjugated to aligand attached at the 3′-terminus, and wherein the ligand is one ormore GalNAc derivatives attached through a bivalent or trivalentbranched linker.

In another aspect, the present invention provides double stranded RNAiagents for inhibiting expression of hepatitis D virus (HDV) in a cell.The double stranded RNAi agents include a sense strand and an antisensestrand forming a double-stranded region, wherein the sense strandcomprises at least 15 contiguous nucleotides differing by no more than 3nucleotides from the nucleotide sequence of SEQ ID NO:31, and theantisense strand comprises at least 15 contiguous nucleotides differingby no more than 3 nucleotides from the nucleotide sequence of SEQ IDNO:32, wherein substantially all of the nucleotides of the sense strandand substantially all of the nucleotides of the antisense strand aremodified nucleotides, wherein the sense strand is conjugated to a ligandattached at the 3′-terminus, and wherein the ligand is one or moreGalNAc derivatives attached through a bivalent or trivalent branchedlinker.

In one aspect, the present invention provides double stranded RNAiagents for inhibiting expression of hepatitis D virus (HDV) in a cell.The double stranded RNAi agents include a sense strand and an antisensestrand forming a double-stranded region, wherein the sense strandcomprises at least 15 contiguous nucleotides differing by no more than 3nucleotides from the nucleotide sequence of SEQ ID NO:33, and theantisense strand comprises at least 15 contiguous nucleotides differingby no more than 3 nucleotides from the nucleotide sequence of SEQ IDNO:34, wherein substantially all of the nucleotides of the sense strandand substantially all of the nucleotides of the antisense strand aremodified nucleotides, wherein the sense strand is conjugated to a ligandattached at the 3′-terminus, and wherein the ligand is one or moreGalNAc derivatives attached through a bivalent or trivalent branchedlinker.

In one aspect, the present invention provides double stranded RNAiagents for inhibiting expression of hepatitis D virus (HDV) in a cell.The double stranded RNAi agents include a sense strand and an antisensestrand forming a double-stranded region, wherein the sense strandcomprises at least 15 contiguous nucleotides differing by no more than 3nucleotides from the nucleotide sequence of SEQ ID NO:35, and theantisense strand comprises at least 15 contiguous nucleotides differingby no more than 3 nucleotides from the nucleotide sequence of SEQ IDNO:36, wherein substantially all of the nucleotides of the sense strandand substantially all of the nucleotides of the antisense strand aremodified nucleotides, wherein the sense strand is conjugated to a ligandattached at the 3′-terminus, and wherein the ligand is one or moreGalNAc derivatives attached through a bivalent or trivalent branchedlinker.

In another aspect, the present invention provides double stranded RNAiagents for inhibiting expression of hepatitis D virus (HDV) in a cell.The double stranded RNAi agents include a sense strand and an antisensestrand forming a double-stranded region, wherein the sense strandcomprises at least 15 contiguous nucleotides differing by no more than 3nucleotides from the nucleotide sequence of SEQ ID NO:37, and theantisense strand comprises at least 15 contiguous nucleotides differingby no more than 3 nucleotides from the nucleotide sequence of SEQ IDNO:38, wherein substantially all of the nucleotides of the sense strandand substantially all of the nucleotides of the antisense strand aremodified nucleotides, wherein the sense strand is conjugated to a ligandattached at the 3′-terminus, and wherein the ligand is one or moreGalNAc derivatives attached through a bivalent or trivalent branchedlinker.

In yet another aspect, the present invention provides double strandedRNAi agents for inhibiting expression of hepatitis D virus (HDV) in acell. The double stranded RNAi agents include a sense strand and anantisense strand forming a double-stranded region, wherein the sensestrand comprises at least 15 contiguous nucleotides differing by no morethan 3 nucleotides from the nucleotide sequence of SEQ ID NO:39, and theantisense strand comprises at least 15 contiguous nucleotides differingby no more than 3 nucleotides from the nucleotide sequence of SEQ IDNO:40, wherein substantially all of the nucleotides of the sense strandand substantially all of the nucleotides of the antisense strand aremodified nucleotides, wherein the sense strand is conjugated to a ligandattached at the 3′-terminus, and wherein the ligand is one or moreGalNAc derivatives attached through a bivalent or trivalent branchedlinker.

In one aspect, the present invention provides double stranded RNAiagents for inhibiting expression of hepatitis D virus (HDV) in a cell.The double stranded RNAi agents include a sense strand and an antisensestrand forming a double-stranded region, wherein the sense strandcomprises at least 15 contiguous nucleotides differing by no more than 3nucleotides from the nucleotide sequence of SEQ ID NO:41, and theantisense strand comprises at least 15 contiguous nucleotides differingby no more than 3 nucleotides from the nucleotide sequence of SEQ IDNO:42, wherein substantially all of the nucleotides of the sense strandand substantially all of the nucleotides of the antisense strand aremodified nucleotides, wherein the sense strand is conjugated to a ligandattached at the 3′-terminus, and wherein the ligand is one or moreGalNAc derivatives attached through a bivalent or trivalent branchedlinker.

In another aspect, the present invention provides double stranded RNAiagents for inhibiting expression of hepatitis D virus (HDV) in a cell.The double stranded RNAi agents include a sense strand and an antisensestrand forming a double-stranded region, wherein the sense strandcomprises at least 15 contiguous nucleotides differing by no more than 3nucleotides from the nucleotide sequence of SEQ ID NO:43, and theantisense strand comprises at least 15 contiguous nucleotides differingby no more than 3 nucleotides from the nucleotide sequence of SEQ IDNO:44, wherein substantially all of the nucleotides of the sense strandand substantially all of the nucleotides of the antisense strand aremodified nucleotides, wherein the sense strand is conjugated to a ligandattached at the 3′-terminus, and wherein the ligand is one or moreGalNAc derivatives attached through a bivalent or trivalent branchedlinker.

In one aspect, the present invention provides double stranded RNAiagents for inhibiting expression of hepatitis D virus (HDV) in a cell.The double stranded RNAi agents include a sense strand and an antisensestrand forming a double-stranded region, wherein the sense strandcomprises at least 15 contiguous nucleotides differing by no more than 3nucleotides from the nucleotide sequence of SEQ ID NO:2551, and theantisense strand comprises at least 15 contiguous nucleotides differingby no more than 3 nucleotides from the nucleotide sequence of SEQ ID NO:2552, wherein substantially all of the nucleotides of the sense strandand substantially all of the nucleotides of the antisense strand aremodified nucleotides, wherein the sense strand is conjugated to a ligandattached at the 3′-terminus, and wherein the ligand is one or moreGalNAc derivatives attached through a bivalent or trivalent branchedlinker. In certain embodiments, the sense strand of the double strandedRNAi agents comprises at least 15 contiguous nucleotides differing by nomore than 3 nucleotides from nucleotides 1451-1484, nucleotides1455-1480, nucleotides 1455-1474, or nucleotides 1417-1443 of thenucleotide sequence of SEQ ID NO:2551.

In one embodiment, the one or more of the 3 nucleotide differences inthe nucleotide sequence of the antisense strand is a nucleotide mismatchin the antisense strand.

In another embodiment, the one or more of the 3 nucleotide differencesin the nucleotide sequence of the antisense strand is a nucleotidemismatch in the sense strand.

In one embodiment, all of the nucleotides of the sense strand and all ofthe nucleotides of the antisense strand are modified nucleotides.

In one embodiment, the sense strand and the antisense strand comprise aregion of complementarity which comprises at least 15 contiguousnucleotides differing by no more than 3 nucleotides from any one of thesequences listed in any one of Tables 11, 12, 31, and 32.

In some embodiments, the sense strand and the antisense strand comprisea region of complementarity which comprises at least 15 contiguousnucleotides differing by no more than 3 nucleotides from the nucleotidesequences of the sense strand and the antisense strand sequences of anyone of duplexes AD-70260.1, AD-70232.1, AD-70249.1, AD-70244.1,AD-70272.1, AD-70228.1, AD-70255.1, AD-70278.1, AD-70295.1, AD-67200.1,AD-67211.1, AD-67199.1, AD-67202.1, AD-67208.1, AD-67210.1, AD-70259.1,AD-70267.1, AD-70272.1, AD-70271.1, AD-70268.1, AD-70269.1, AD-70232.1,AD-70256.1, AD-70257.1, and AD-70275.1. In other embodiments, the sensestrand and the antisense strand comprise a region of complementaritywhich comprises at least 15 contiguous nucleotides differing by no morethan 3 nucleotides from the nucleotide sequences of the sense strand andthe antisense strand sequences of any one of duplexes AD-70260.1,AD-70232.1, AD-70249.1, AD-70244.1, AD-70272.1, AD-70228.1, AD-70255.1,AD-70278.1, and AD-70295.1.

In one embodiment, the at least one of the modified nucleotides isselected from the group consisting of a 3′-terminal deoxy-thymine (dT)nucleotide, a 2′-O-methyl modified nucleotide, a 2′-fluoro modifiednucleotide, a 2′-deoxy-modified nucleotide, a locked nucleotide, anunlocked nucleotide, a conformationally restricted nucleotide, aconstrained ethyl nucleotide, an abasic nucleotide, a 2′-amino-modifiednucleotide, a 2′-O-allyl-modified nucleotide, 2′-C-alkyl-modifiednucleotide, 2′-hydroxyl-modified nucleotide, a 2′-methoxyethyl modifiednucleotide, a 2′-O-alkyl-modified nucleotide, a morpholino nucleotide, aphosphoramidate, a non-natural base comprising nucleotide, atetrahydropyran modified nucleotide, a 1,5-anhydrohexitol modifiednucleotide, a cyclohexenyl modified nucleotide, a nucleotide comprisinga phosphorothioate group, a nucleotide comprising a methylphosphonategroup, a nucleotide comprising a 5′-phosphate, and a nucleotidecomprising a 5′-phosphate mimic.

In one embodiment, the at least one strand comprises a 3′ overhang of atleast 1 nucleotide. In another embodiment, the at least one strandcomprises a 3′ overhang of at least 2 nucleotides.

In one embodiment, the double-stranded region is 15-30 nucleotide pairsin length. In another embodiment, the double-stranded region is 17-23nucleotide pairs in length. In yet another embodiment, thedouble-stranded region is 17-25 nucleotide pairs in length. In oneembodiment, the e double-stranded region is 23-27 nucleotide pairs inlength. In another embodiment, the double-stranded region is 19-21nucleotide pairs in length. In yet another embodiment, thedouble-stranded region is 21-23 nucleotide pairs in length.

In one embodiment, each strand has 15-30 nucleotides. In anotherembodiment, each strand has 19-30 nucleotides.

In one embodiment, the ligand is

In one embodiment, the RNAi agent is conjugated to the ligand as shownin the following schematic

wherein X is O or S.

In one embodiment, the RNAi agent is selected from the group of RNAiagents listed in any one of Tables 11, 12, 31, and 32.

In one embodiment the agent is selected from the group consisting ofAD-70260.1, AD-70232.1, AD-70249.1, AD-70244.1, AD-70272.1, AD-70228.1,AD-70255.1, AD-70278.1, AD-70295.1, AD-67200.1, AD-67211.1, AD-67199.1,AD-67202.1, AD-67208.1, AD-67210.1, AD-70259.1, AD-70267.1, AD-70272.1,AD-70271.1, AD-70268.1, AD-70269.1, AD-70232.1, AD-70256.1, AD-70257.1,and AD-70275.1.

In one aspect, the present invention provides double stranded RNAiagents for inhibiting expression of hepatitis D virus (HDV) in a cell.The double stranded RNAi agents include a sense strand and an antisensestrand forming a double-stranded region, wherein the sense strandcomprises any one of the sense sequences from Tables 11, 12, 31, and 32,and the antisense strand comprises any one of the antisense sequencesfrom Tables 11, 12, 31, and 32, wherein substantially all of thenucleotides of the sense strand and substantially all of the nucleotidesof the antisense strand are modified nucleotides, wherein the sensestrand is conjugated to a ligand attached at the 3′-terminus, andwherein the ligand is one or more GalNAc derivatives attached through abivalent or trivalent branched linker.

In one embodiment, the sense strand is selected from the groupconsisting of the sense strand sequences of AD-70260.1, AD-70232.1,AD-70249.1, AD-70244.1, AD-70272.1, AD-70228.1, AD-70255.1, AD-70278.1,AD-70295.1, AD-67200.1, AD-67211.1, AD-67199.1, AD-67202.1, AD-67208.1,AD-67210.1, AD-70259.1, AD-70267.1, AD-70272.1, AD-70271.1, AD-70268.1,AD-70269.1, AD-70232.1, AD-70256.1, AD-70257.1, or AD-70275.1.

In another embodiment, the antisense strand is selected from the groupconsisting of the antisense strand sequences of AD-70260.1, AD-70232.1,AD-70249.1, AD-70244.1, AD-70272.1, AD-70228.1, AD-70255.1, AD-70278.1,AD-70295.1, AD-67200.1, AD-67211.1, AD-67199.1, AD-67202.1, AD-67208.1,AD-67210.1, AD-70259.1, AD-70267.1, AD-70272.1, AD-70271.1, AD-70268.1,AD-70269.1, AD-70232.1, AD-70256.1, AD-70257.1, or AD-70275.1.

The present invention also provides RNAi agents comprising sense andantisense nucleotide sequences which are at least 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, or 99% identical over their entire length tothe foregoing sense and antisense nucleotide sequences

In one embodiment, all of the nucleotides of the sense strand and all ofthe nucleotides of the antisense strand comprise a modification.

In one embodiment, at least one of the modified nucleotides is selectedfrom the group consisting of a 3′-terminal deoxy-thymine (dT)nucleotide, a 2′-O-methyl modified nucleotide, a 2′-fluoro modifiednucleotide, a 2′-deoxy-modified nucleotide, a locked nucleotide, anunlocked nucleotide, a conformationally restricted nucleotide, aconstrained ethyl nucleotide, an abasic nucleotide, a 2′-amino-modifiednucleotide, a 2′-O-allyl-modified nucleotide, 2′-C-alkyl-modifiednucleotide, 2′-hydroxyl-modified nucleotide, a 2′-methoxyethyl modifiednucleotide, a 2′-O-alkyl-modified nucleotide, a morpholino nucleotide, aphosphoramidate, a non-natural base comprising nucleotide, atetrahydropyran modified nucleotide, a 1,5-anhydrohexitol modifiednucleotide, a cyclohexenyl modified nucleotide, a nucleotide comprisinga phosphorothioate group, a nucleotide comprising a methylphosphonategroup, a nucleotide comprising a 5′-phosphate, and a nucleotidecomprising a 5′-phosphate mimic.

In one embodiment, the 5′-phosphate mimic is a 5′-vinyl phosphate(5′-VP).

In one embodiment, the ligand is

In one embodiment, the RNAi agent is conjugated to the ligand as shownin the following schematic

wherein X is O or S.

In one aspect, the present invention provides compositions comprisingtwo or more double stranded RNAi agents for inhibiting expression ofhepatitis D virus (HDV) in a cell, wherein each double stranded RNAiagent independently comprises a sense strand and an antisense strandforming a double-stranded region, wherein each of the sense strandsindependently comprises at least 15 contiguous nucleotides differing byno more than 3 nucleotides from the nucleotide sequence of SEQ ID NO:29,and each of the antisense strands independently comprises at least 15contiguous nucleotides differing by no more than 3 nucleotides from thenucleotide sequence of SEQ ID NO:30, wherein substantially all of thenucleotides of each of the sense strands and substantially all of thenucleotides of each of the antisense strands are independently modifiednucleotides, wherein each of the sense strands are independentlyconjugated to a ligand attached at the 3′-terminus, and wherein theligand is one or more GalNAc derivatives attached through a bivalent ortrivalent branched linker.

In another aspect, the present invention provides compositionscomprising two or more double stranded RNAi agents for inhibitingexpression of hepatitis D virus (HDV) in a cell, wherein each doublestranded RNAi agent independently comprises a sense strand and anantisense strand forming a double-stranded region, wherein each of thesense strands independently comprises at least 15 contiguous nucleotidesdiffering by no more than 3 nucleotides from the nucleotide sequence ofSEQ ID NO:31, and each of the antisense strands independently comprisesat least 15 contiguous nucleotides differing by no more than 3nucleotides from the nucleotide sequence of SEQ ID NO:32, whereinsubstantially all of the nucleotides of each of the sense strands andsubstantially all of the nucleotides of each of the antisense strandsare independently modified nucleotides, wherein each of the sensestrands are independently conjugated to a ligand attached at the3′-terminus, and wherein the ligand is one or more GalNAc derivativesattached through a bivalent or trivalent branched linker.

In another aspect, the present invention provides compositionscomprising two or more double stranded RNAi agents for inhibitingexpression of hepatitis D virus (HDV) in a cell, wherein each doublestranded RNAi agent independently comprises a sense strand and anantisense strand forming a double-stranded region, wherein each of thesense strands independently comprises at least 15 contiguous nucleotidesdiffering by no more than 3 nucleotides from the nucleotide sequence ofSEQ ID NO:33, and each of the antisense strands independently comprisesat least 15 contiguous nucleotides differing by no more than 3nucleotides from the nucleotide sequence of SEQ ID NO:34, whereinsubstantially all of the nucleotides of each of the sense strands andsubstantially all of the nucleotides of each of the antisense strandsare independently modified nucleotides, wherein each of the sensestrands are independently conjugated to a ligand attached at the3′-terminus, and wherein the ligand is one or more GalNAc derivativesattached through a bivalent or trivalent branched linker.

In yet another aspect, the present invention provides compositionscomprising two or more double stranded RNAi agents for inhibitingexpression of hepatitis D virus (HDV) in a cell, wherein each doublestranded RNAi agent independently comprises a sense strand and anantisense strand forming a double-stranded region, wherein each of thesense strands independently comprises at least 15 contiguous nucleotidesdiffering by no more than 3 nucleotides from the nucleotide sequence ofSEQ ID NO:35, and each of the antisense strands independently comprisesat least 15 contiguous nucleotides differing by no more than 3nucleotides from the nucleotide sequence of SEQ ID NO:36, whereinsubstantially all of the nucleotides of each of the sense strands andsubstantially all of the nucleotides of each of the antisense strandsare independently modified nucleotides, wherein each of the sensestrands are independently conjugated to a ligand attached at the3′-terminus, and wherein the ligand is one or more GalNAc derivativesattached through a bivalent or trivalent branched linker.

In one aspect, the present invention provides compositions comprisingtwo or more double stranded RNAi agents for inhibiting expression ofhepatitis D virus (HDV) in a cell, wherein each double stranded RNAiagent independently comprises a sense strand and an antisense strandforming a double-stranded region, wherein each of the sense strandsindependently comprises at least 15 contiguous nucleotides differing byno more than 3 nucleotides from the nucleotide sequence of SEQ ID NO:37,and each of the antisense strands independently comprises at least 15contiguous nucleotides differing by no more than 3 nucleotides from thenucleotide sequence of SEQ ID NO:38, wherein substantially all of thenucleotides of each of the sense strands and substantially all of thenucleotides of each of the antisense strands are independently modifiednucleotides, wherein each of the sense strands are independentlyconjugated to a ligand attached at the 3′-terminus, and wherein theligand is one or more GalNAc derivatives attached through a bivalent ortrivalent branched linker.

In another aspect, the present invention provides compositionscomprising two or more double stranded RNAi agents for inhibitingexpression of hepatitis D virus (HDV) in a cell, wherein each doublestranded RNAi agent independently comprises a sense strand and anantisense strand forming a double-stranded region, wherein each of thesense strands independently comprises at least 15 contiguous nucleotidesdiffering by no more than 3 nucleotides from the nucleotide sequence ofSEQ ID NO:39, and each of the antisense strands independently comprisesat least 15 contiguous nucleotides differing by no more than 3nucleotides from the nucleotide sequence of SEQ ID NO:40, whereinsubstantially all of the nucleotides of each of the sense strands andsubstantially all of the nucleotides of each of the antisense strandsare independently modified nucleotides, wherein each of the sensestrands are independently conjugated to a ligand attached at the3′-terminus, and wherein the ligand is one or more GalNAc derivativesattached through a bivalent or trivalent branched linker.

In another aspect, the present invention provides compositionscomprising two or more double stranded RNAi agents for inhibitingexpression of hepatitis D virus (HDV) in a cell, wherein each doublestranded RNAi agent independently comprises a sense strand and anantisense strand forming a double-stranded region, wherein each of thesense strands independently comprises at least 15 contiguous nucleotidesdiffering by no more than 3 nucleotides from the nucleotide sequence ofSEQ ID NO:41, and each of the antisense strands independently comprisesat least 15 contiguous nucleotides differing by no more than 3nucleotides from the nucleotide sequence of SEQ ID NO:42, whereinsubstantially all of the nucleotides of each of the sense strands andsubstantially all of the nucleotides of each of the antisense strandsare independently modified nucleotides, wherein each of the sensestrands are independently conjugated to a ligand attached at the3′-terminus, and wherein the ligand is one or more GalNAc derivativesattached through a bivalent or trivalent branched linker.

In one aspect, the present invention provides compositions comprisingtwo or more double stranded RNAi agents for inhibiting expression ofhepatitis D virus (HDV) in a cell, wherein each double stranded RNAiagent independently comprises a sense strand and an antisense strandforming a double-stranded region, wherein each of the sense strandsindependently comprises at least 15 contiguous nucleotides differing byno more than 3 nucleotides from the nucleotide sequence of SEQ ID NO:43,and each of the antisense strands independently comprises at least 15contiguous nucleotides differing by no more than 3 nucleotides from thenucleotide sequence of SEQ ID NO:44, wherein substantially all of thenucleotides of each of the sense strands and substantially all of thenucleotides of each of the antisense strands are independently modifiednucleotides, wherein each of the sense strands are independentlyconjugated to a ligand attached at the 3′-terminus, and wherein theligand is one or more GalNAc derivatives attached through a bivalent ortrivalent branched linker.

In another aspect, the present invention provides compositionscomprising two or more double stranded RNAi agents for inhibitingexpression of hepatitis D virus (HDV) in a cell, wherein each doublestranded RNAi agent independently comprises a sense strand and anantisense strand forming a double-stranded region, wherein each of thesense strands independently comprises at least 15 contiguous nucleotidesdiffering by no more than 3 nucleotides from the nucleotide sequence ofSEQ ID NO:2551, and each of the antisense strands independentlycomprises at least 15 contiguous nucleotides differing by no more than 3nucleotides from the nucleotide sequence of SEQ ID NO:2552, whereinsubstantially all of the nucleotides of each of the sense strands andsubstantially all of the nucleotides of each of the antisense strandsare independently modified nucleotides, wherein each of the sensestrands are independently conjugated to a ligand attached at the3′-terminus, and wherein the ligand is one or more GalNAc derivativesattached through a bivalent or trivalent branched linker.

In certain embodiments, the sense strand of one or both of the first andsecond double stranded RNAi agents comprises at least 15 contiguousnucleotides from nucleotides 1451-1484, nucleotides 1455-1480,nucleotides 1455-1474, or nucleotides 1417-1443 of the nucleotidesequence of SEQ ID NO:2551.

In one embodiment, the one or more of the 3 nucleotide differences inthe nucleotide sequence of the antisense strand is a nucleotide mismatchin the antisense strand. In another embodiment, the one or more of the 3nucleotide differences in the nucleotide sequence of the antisensestrand is a nucleotide mismatch in the sense strand.

In one embodiment, all of the nucleotides of the sense strand and all ofthe nucleotides of the antisense strand are modified nucleotides.

In one embodiment, the sense strand and the antisense strand comprise aregion of complementarity which comprises at least 15 contiguousnucleotides differing by no more than 3 nucleotides from any one of thesequences listed in any one of Tables 11, 12, 31, and 32.

In some embodiments, the sense strand and the antisense strand comprisea region of complementarity which comprises at least 15 contiguousnucleotides differing by no more than 3 nucleotides from the nucleotidesequences of the sense strand and the antisense strand sequences of anyone of duplexes AD-70260.1, AD-70232.1, AD-70249.1, AD-70244.1,AD-70272.1, AD-70228.1, AD-70255.1, AD-70278.1, AD-70295.1, AD-67200.1,AD-67211.1, AD-67199.1, AD-67202.1, AD-67208.1, AD-67210.1, AD-70259.1,AD-70267.1, AD-70272.1, AD-70271.1, AD-70268.1, AD-70269.1, AD-70232.1,AD-70256.1, AD-70257.1, or AD-70275.1.

In other embodiments, the sense strand and the antisense strand comprisea region of complementarity which comprises at least 15 contiguousnucleotides differing by no more than 3 nucleotides from the nucleotidesequences of the sense strand and antisense strand sequences of any oneof duplexes AD-70260.1, AD-70232.1, AD-70249.1, AD-70244.1, AD-70272.1,AD-70228.1, AD-70255.1, AD-70278.1, or AD-70295.1.

In one embodiment, the at least one of the modified nucleotides isselected from the group consisting of a 3′-terminal deoxy-thymine (dT)nucleotide, a 2′-O-methyl modified nucleotide, a 2′-fluoro modifiednucleotide, a 2′-deoxy-modified nucleotide, a locked nucleotide, anunlocked nucleotide, a conformationally restricted nucleotide, aconstrained ethyl nucleotide, an abasic nucleotide, a 2′-amino-modifiednucleotide, a 2′-O-allyl-modified nucleotide, 2′-C-alkyl-modifiednucleotide, 2′-hydroxyl-modified nucleotide, a 2′-methoxyethyl modifiednucleotide, a 2′-O-alkyl-modified nucleotide, a morpholino nucleotide, aphosphoramidate, a non-natural base comprising nucleotide, atetrahydropyran modified nucleotide, a 1,5-anhydrohexitol modifiednucleotide, a cyclohexenyl modified nucleotide, a nucleotide comprisinga phosphorothioate group, a nucleotide comprising a methylphosphonategroup, a nucleotide comprising a 5′-phosphate, and a nucleotidecomprising a 5′-phosphate mimic.

In another aspect, the present invention provides compositions forinhibiting expression of hepatitis D virus (HDV) in a cell, thecomposition comprising (a) a first double-stranded RNAi agent comprisinga first sense strand and a first antisense strand forming adouble-stranded region, wherein substantially all of the nucleotides ofthe first sense strand and substantially all of the nucleotides of thefirst antisense strand are modified nucleotides, wherein the first sensestrand is conjugated to a ligand attached at the 3′-terminus, andwherein the ligand is one or more GalNAc derivatives attached through abivalent or trivalent branched linker; and (b) a second double-strandedRNAi agent comprising a second sense strand and a second antisensestrand forming a double-stranded region, wherein substantially all ofthe nucleotides of the second sense strand and substantially all of thenucleotides of the second antisense strand are modified nucleotides,wherein the second sense strand is conjugated to a ligand attached atthe 3′-terminus, and wherein the ligand is one or more GalNAcderivatives attached through a bivalent or trivalent branched linker;wherein the first and second sense strands each independently comprise asequence selected from the group consisting of any one of the sensesequences from Tables 11, 12, 31, and 32 (or a nucleotide sequence whichis at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical over its entire length to the foregoing nucleotide sequences),and wherein the first and second antisense strands each independentlycomprise a sequence selected from the group consisting of any one of theantisense sequences from Tables 11, 12, 31, and 32 (or a nucleotidesequence which is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,or 99% identical over its entire length to the foregoing nucleotidesequences).

In one embodiment, the first and second sense strands each independentlycomprise a sequence selected from the group consisting of any one of thesense sequences from AD-70260.1, AD-70232.1, AD-70249.1, AD-70244.1,AD-70272.1, AD-70228.1, AD-70255.1, AD-70278.1, AD-70295.1, AD-67200.1,AD-67211.1, AD-67199.1, AD-67202.1, AD-67208.1, AD-67210.1, AD-70259.1,AD-70267.1, AD-70272.1, AD-70271.1, AD-70268.1, AD-70269.1, AD-70232.1,AD-70256.1, AD-70257.1, or AD-70275.1 (or a nucleotide sequence which isat least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identicalover its entire length to the foregoing nucleotide sequences).

In another embodiment, the first and second antisense strands eachindependently comprise a sequence selected from the group consisting ofany one of the antisense sequences from AD-70260.1, AD-70232.1,AD-70249.1, AD-70244.1, AD-70272.1, AD-70228.1, AD-70255.1, AD-70278.1,AD-70295.1, AD-67200.1, AD-67211.1, AD-67199.1, AD-67202.1, AD-67208.1,AD-67210.1, AD-70259.1, AD-70267.1, AD-70272.1, AD-70271.1, AD-70268.1,AD-70269.1, AD-70232.1, AD-70256.1, AD-70257.1, or AD-70275.1 (or anucleotide sequence which is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, or 99% identical over its entire length to the foregoingnucleotide sequences).

In one embodiment, the first and second sense strand and/or all of thenucleotides of the first and second antisense strand comprise amodification.

In one embodiment, the at least one of the modified nucleotides isselected from the group consisting of a 3′-terminal deoxy-thymine (dT)nucleotide, a 2′-O-methyl modified nucleotide, a 2′-fluoro modifiednucleotide, a 2′-deoxy-modified nucleotide, a locked nucleotide, anunlocked nucleotide, a conformationally restricted nucleotide, aconstrained ethyl nucleotide, an abasic nucleotide, a 2′-amino-modifiednucleotide, a 2′-O-allyl-modified nucleotide, 2′-C-alkyl-modifiednucleotide, 2′-hydroxyl-modified nucleotide, a 2′-methoxyethyl modifiednucleotide, a 2′-O-alkyl-modified nucleotide, a morpholino nucleotide, aphosphoramidate, a non-natural base comprising nucleotide, atetrahydropyran modified nucleotide, a 1,5-anhydrohexitol modifiednucleotide, a cyclohexenyl modified nucleotide, a nucleotide comprisinga phosphorothioate group, a nucleotide comprising a methylphosphonategroup, a nucleotide comprising a 5′-phosphate, and a nucleotidecomprising a 5′-phosphate mimic.

In one embodiment, the first and second RNAi agent are selected from thegroup consisting of any one of the the sequences provided in Tables 11,12, 31, and 32.

In one embodiment, the first and second RNAi agent are selected from thegroup consisting of any one of AD-70260.1, AD-70232.1, AD-70249.1,AD-70244.1, AD-70272.1, AD-70228.1, AD-70255.1, AD-70278.1, AD-70295.1,AD-67200.1, AD-67211.1, AD-67199.1, AD-67202.1, AD-67208.1, AD-67210.1,AD-70259.1, AD-70267.1, AD-70272.1, AD-70271.1, AD-70268.1, AD-70269.1,AD-70232.1, AD-70256.1, AD-70257.1, or AD-70275.1.

In one embodiment, the first and second RNAi agent are selected from thegroup consisting of any one of AD-70260.1, AD-70232.1, AD-70249.1,AD-70244.1, AD-70272.1, AD-70228.1, AD-70255.1, AD-70278.1, orAD-70295.1.

In one aspect, the present invention provides a double stranded RNAiagent comprising any one of the RNAi agents listed in any one of Tables11, 12, 31, 32,

In one embodiment the agent is selected from the group consisting ofAD-70260.1, AD-70232.1, AD-70249.1, AD-70244.1, AD-70272.1, AD-70228.1,AD-70255.1, AD-70278.1, AD-70295.1, AD-67200.1, AD-67211.1, AD-67199.1,AD-67202.1, AD-67208.1, AD-67210.1, AD-70259.1, AD-70267.1, AD-70272.1,AD-70271.1, AD-70268.1, AD-70269.1, AD-70232.1, AD-70256.1, AD-70257.1,or AD-70275.1. In another embodiment, the agent is selected from thegroup consisting of AD-70260.1, AD-70232.1, AD-70249.1, AD-70244.1,AD-70272.1, AD-70228.1, AD-70255.1, AD-70278.1, or AD-70295.1.

The present invention also provides vectors and cells comprising thedouble stranded RNAi agent of the invention.

In another aspect, the present invention provides pharmaceuticalcompositions comprising the double stranded RNAi agents of theinvention, or the compositions of the invention, or the vectors of theinvention.

In one embodiment, the double stranded RNAi agent is administered in anunbuffered solution. In one embodiment, the unbuffered solution issaline or water.

In another embodiment, the double stranded RNAi agent is administeredwith a buffer solution. In one embodiment, the buffer solution comprisesacetate, citrate, prolamine, carbonate, or phosphate or any combinationthereof. In another embodiment, the buffer solution is phosphatebuffered saline (PBS).

In one aspect, the present invention provides methods of inhibitingHepatitis D virus (HDV) gene expression in a cell. The methods includecontacting the cell with the double stranded RNAi agent of theinvention, or the composition of the invention, or the vector of theinvention, or the pharmaceutical composition of the invention; andmaintaining the cell produced for a time sufficient to obtaindegradation of the mRNA transcript of an HDV gene, thereby inhibitingexpression of the HDV gene in the cell.

In one aspect, the present invention provides methods of inhibitingreplication of a Hepatitis D virus (HDV) in a cell. The methods includecontacting the cell with the double stranded RNAi agent of theinvention, or the composition of the invention, or the vector of theinvention, or the pharmaceutical composition of the invention; andmaintaining the cell produced for a time sufficient to obtaindegradation of the mRNA transcript of an HDV gene, thereby inhibitingreplication of the HDV in the cell.

In one embodiment, the cell is within a subject. In one embodiment, thesubject is a human.

In one embodiment, the subject suffers from an HDV-associated disease.

In one embodiment, HDV gene expression is inhibited by at least about30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%,about 95%, about 98% or about 100%.

In one embodiment, replication of HDV in the cell is inhibited by atleast about 30%, about 40%, about 50%, about 60%, about 70%, about 80%,about 90%, about 95%, about 98% or about 100%.

In one aspect, the present invention provides methods of reducing thelevel of Hepatitis D virus (HDV) DNA in a subject infected with HDV. Themethods include administering to the subject a therapeutically effectiveamount of the double stranded RNAi agent of the invention, or thecomposition of the invention, or the vector of the invention, or thepharmaceutical composition of the invention, thereby reducing the levelof HDV DNA in the subject.

In another aspect, the present invention provides methods of reducingthe level of a Hepatitis D virus (HDV) antigen in a subject infectedwith HDV. The methods include administering to the subject atherapeutically effective amount of the double stranded RNAi agent ofthe invention, or the composition of the invention, or the vector of theinvention, or the pharmaceutical composition of the invention, therebyreducing the level of the HDV antigen in the subject. The level of thelarge HDV antigen may be reduced, the level of the small HDV antigen maybe reduced, or the level of both the large and the small HDV antigensmay be reduced.

In another aspect, the present invention provides methods of reducingthe viral load of Hepatitis D virus (HDV) in a subject infected withHDV. The methods include administering to the subject a therapeuticallyeffective amount of the double stranded RNAi agent of the invention, orthe composition of the invention, or the vector of the invention, or thepharmaceutical composition of the invention, thereby reducing the viralload of HDV in the subject.

In one aspect, the present invention provides methods of treating asubject having a Hepatitis D virus (HDV) infection. The methods includeadministering to the subject a therapeutically effective amount of thedouble stranded RNAi agent of the invention, or the composition of theinvention, or the vector of the invention, or the pharmaceuticalcomposition of the invention, thereby treating the subject.

In another aspect, the present invention provides methods of treating asubject having a Hepatitis D virus (HDV)-associated disorder. Themethods include administering to the subject a therapeutically effectiveamount of the double stranded RNAi agent of the invention, or thecomposition of the invention, or the vector of the invention, or thepharmaceutical composition of the invention, thereby treating thesubject.

In one embodiment, the HDV-associated disorder is selected from thegroup consisting of hepatitis B virus infection, acute hepatitis B,acute hepatitis D; acute fulminant hepatitis D; chronic hepatitis D;liver fibrosis; end-stage liver disease; hepatocellular carcinoma.

In one aspect, the present invention provides methods of treating asubject having a Hepatitis D virus (HDV) infection. The methods includeadministering to the subject a therapeutically effective amount of adouble stranded RNAi agent, wherein the double stranded RNAi agentcomprises a sense strand and an antisense strand forming adouble-stranded region, wherein the sense strand comprises any one sensesequences from any one of Tables 11, 12, 31, and 32 (or a nucleotidesequence which is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,or 99% identical over its entire length to the foregoing nucleotidesequence), and the antisense strand comprises any one of the antisensesequences from any one of Tables 11, 12, 31, and 32 (or a nucleotidesequence which is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,or 99% identical over its entire length to the foregoing nucleotidesequence), wherein substantially all of the nucleotides of the sensestrand and substantially all of the nucleotides of the antisense strandare modified nucleotides, wherein the sense strand is conjugated to aligand attached at the 3′-terminus, and wherein the ligand is one ormore GalNAc derivatives attached through a bivalent or trivalentbranched linker, thereby treating the subject.

In one embodiment, the sense strand comprises a sequence selected fromthe group consisting of any one of the sense sequences from AD-70260.1,AD-70232.1, AD-70249.1, AD-70244.1, AD-70272.1, AD-70228.1, AD-70255.1,AD-70278.1, AD-70295.1, AD-67200.1, AD-67211.1, AD-67199.1, AD-67202.1,AD-67208.1, AD-67210.1, AD-70259.1, AD-70267.1, AD-70272.1, AD-70271.1,AD-70268.1, AD-70269.1, AD-70232.1, AD-70256.1, AD-70257.1, orAD-70275.1 (or a nucleotide sequence which is at least 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or 99% identical over its entire length tothe foregoing nucleotide sequences).

In another embodiment, the antisense strand comprises a sequenceselected from the group consisting of any one of the antisense sequencesfrom AD-70260.1, AD-70232.1, AD-70249.1, AD-70244.1, AD-70272.1,AD-70228.1, AD-70255.1, AD-70278.1, AD-70295.1, AD-67200.1, AD-67211.1,AD-67199.1, AD-67202.1, AD-67208.1, AD-67210.1, AD-70259.1, AD-70267.1,AD-70272.1, AD-70271.1, AD-70268.1, AD-70269.1, AD-70232.1, AD-70256.1,AD-70257.1, or AD-70275.1 (or a nucleotide sequence which is at least90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical over itsentire length to the foregoing nucleotide sequences).

In one embodiment, all of the nucleotides of the sense strand and all ofthe nucleotides of the antisense strand comprise a modification.

In one embodiment, the at least one of the modified nucleotides isselected from the group consisting of a 3′-terminal deoxy-thymine (dT)nucleotide, a 2′-O-methyl modified nucleotide, a 2′-fluoro modifiednucleotide, a 2′-deoxy-modified nucleotide, a locked nucleotide, anunlocked nucleotide, a conformationally restricted nucleotide, aconstrained ethyl nucleotide, an abasic nucleotide, a 2′-amino-modifiednucleotide, a 2′-O-allyl-modified nucleotide, 2′-C-alkyl-modifiednucleotide, 2′-hydroxyl-modified nucleotide, a 2′-methoxyethyl modifiednucleotide, a 2′-O-alkyl-modified nucleotide, a morpholino nucleotide, aphosphoramidate, a non-natural base comprising nucleotide, atetrahydropyran modified nucleotide, a 1,5-anhydrohexitol modifiednucleotide, a cyclohexenyl modified nucleotide, a nucleotide comprisinga phosphorothioate group, a nucleotide comprising a methylphosphonategroup, a nucleotide comprising a 5′-phosphate, and a nucleotidecomprising a 5′-phosphate mimic.

In one embodiment, the 5′-phosphate mimic is a 5′-vinyl phosphate(5′-VP).

In one embodiment, the ligand is

In one embodiment, the RNAi agent is conjugated to the ligand as shownin the following schematic

wherein X is O or S.

In one embodiment, the HDV-associated disorder is selected from thegroup consisting of hepatitis B virus infection, acute hepatitis B,acute hepatitis D; acute fulminant hepatitis D; chronic hepatitis D;liver fibrosis; end-stage liver disease; hepatocellular carcinoma.

In one aspect, the present invention provides methods of treating asubject having a Hepatitis D virus (HDV) infection. The methods includeadministering to the subject a therapeutically effective amount of acomposition for inhibiting expression of hepatitis D virus (HDV) in acell. The composition includes: (a) a first double-stranded RNAi agentcomprising a first sense strand and a first antisense strand forming adouble-stranded region, wherein substantially all of the nucleotides ofthe first sense strand and substantially all of the nucleotides of thefirst antisense strand are modified nucleotides, wherein the first sensestrand is conjugated to a ligand attached at the 3′-terminus, andwherein the ligand is one or more GalNAc derivatives attached through abivalent or trivalent branched linker; and (b) a second double-strandedRNAi agent comprising a second sense strand and a second antisensestrand forming a double-stranded region, wherein substantially all ofthe nucleotides of the second sense strand and substantially all of thenucleotides of the second antisense strand are modified nucleotides,wherein the second sense strand is conjugated to a ligand attached atthe 3′-terminus, and wherein the ligand is one or more GalNAcderivatives attached through a bivalent or trivalent branched linker;wherein the first and second sense strands each independently comprise asequence selected from the group consisting of any one of the sensesequences in any one of Tables 11, 12, 31, and 32 (or a nucleotidesequence which is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,or 99% identical over its entire length to any of the foregoingnucleotide sequences), and wherein the first and second antisensestrands each independently comprise a sequence selected from the groupconsisting of any one of the antisense sequences from any one of Tables11, 12, 31, and 32 (or a nucleotide sequence which is at least 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical over its entirelength to any of the foregoing nucleotide sequences), thereby treatingthe subject.

In one embodiment, the first and second sense strands each independentlycomprise a sequence selected from the group consisting of any one of thesense sequences from AD-70260.1, AD-70232.1, AD-70249.1, AD-70244.1,AD-70272.1, AD-70228.1, AD-70255.1, AD-70278.1, AD-70295.1, AD-67200.1,AD-67211.1, AD-67199.1, AD-67202.1, AD-67208.1, AD-67210.1, AD-70259.1,AD-70267.1, AD-70272.1, AD-70271.1, AD-70268.1, AD-70269.1, AD-70232.1,AD-70256.1, AD-70257.1, or AD-70275.1 (or a nucleotide sequence which isat least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identicalover its entire length to the foregoing nucleotide sequences).

In another embodiment, the first and second antisense strands eachindependently comprise a sequence selected from the group consisting ofany one of the antisense sequences from AD-70260.1, AD-70232.1,AD-70249.1, AD-70244.1, AD-70272.1, AD-70228.1, AD-70255.1, AD-70278.1,AD-70295.1, AD-67200.1, AD-67211.1, AD-67199.1, AD-67202.1, AD-67208.1,AD-67210.1, AD-70259.1, AD-70267.1, AD-70272.1, AD-70271.1, AD-70268.1,AD-70269.1, AD-70232.1, AD-70256.1, AD-70257.1, or AD-70275.1 (or anucleotide sequence which is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, or 99% identical over its entire length to the foregoingnucleotide sequences).

In another aspect, the present invention provides methods of treating asubject having a Hepatitis D virus (HDV)-associated disorder. Themethods include administering to the subject a therapeutically effectiveamount of a composition for inhibiting expression of hepatitis D virus(HDV) in a cell. The composition includes: (a) a first double-strandedRNAi agent comprising a first sense strand and a first antisense strandforming a double-stranded region, wherein substantially all of thenucleotides of the first sense strand and substantially all of thenucleotides of the first antisense strand are modified nucleotides,wherein the first sense strand is conjugated to a ligand attached at the3′-terminus, and wherein the ligand is one or more GalNAc derivativesattached through a bivalent or trivalent branched linker; and (b) asecond double-stranded RNAi agent comprising a second sense strand and asecond antisense strand forming a double-stranded region, whereinsubstantially all of the nucleotides of the second sense strand andsubstantially all of the nucleotides of the second antisense strand aremodified nucleotides, wherein the second sense strand is conjugated to aligand attached at the 3′-terminus, and wherein the ligand is one ormore GalNAc derivatives attached through a bivalent or trivalentbranched linker; wherein the first and second sense strands eachindependently comprise a sequence selected from the group consisting ofany one of the sense sequences from any one of Tables 11, 12, 31, and 32(or a nucleotide sequence which is at least 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, or 99% identical over its entire length to any ofthe foregoing nucleotide sequences), and wherein the first and secondantisense strands each independently comprise a sequence selected fromthe group consisting of any one of the antisense sequences from any oneof Tables 11, 12, 31, and 32 (or a nucleotide sequence which is at least90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical over itsentire length to any of the foregoing nucleotide sequences), therebytreating the subject.

In one embodiment, the first and second sense strands each independentlycomprise a sequence selected from the group consisting of any one of thesense sequences from AD-70260.1, AD-70232.1, AD-70249.1, AD-70244.1,AD-70272.1, AD-70228.1, AD-70255.1, AD-70278.1, AD-70295.1, AD-67200.1,AD-67211.1, AD-67199.1, AD-67202.1, AD-67208.1, AD-67210.1, AD-70259.1,AD-70267.1, AD-70272.1, AD-70271.1, AD-70268.1, AD-70269.1, AD-70232.1,AD-70256.1, AD-70257.1, or AD-70275.1 (or a nucleotide sequence which isat least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identicalover its entire length to the foregoing nucleotide sequences).

In another embodiment, the first and second antisense strands eachindependently comprise a sequence selected from the group consisting ofany one of the antisense sequences from AD-70260.1, AD-70232.1,AD-70249.1, AD-70244.1, AD-70272.1, AD-70228.1, AD-70255.1, AD-70278.1,AD-70295.1, AD-67200.1, AD-67211.1, AD-67199.1, AD-67202.1, AD-67208.1,AD-67210.1, AD-70259.1, AD-70267.1, AD-70272.1, AD-70271.1, AD-70268.1,AD-70269.1, AD-70232.1, AD-70256.1, AD-70257.1, or AD-70275.1 (or anucleotide sequence which is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, or 99% identical over its entire length to the foregoingnucleotide sequences).

In one embodiment, all of the nucleotides of the first and second sensestrand and all of the nucleotides of the first and second antisensestrand comprise a modification.

In one embodiment, the at least one of the modified nucleotides isselected from the group consisting of a 3′-terminal deoxy-thymine (dT)nucleotide, a 2′-O-methyl modified nucleotide, a 2′-fluoro modifiednucleotide, a 2′-deoxy-modified nucleotide, a locked nucleotide, anunlocked nucleotide, a conformationally restricted nucleotide, aconstrained ethyl nucleotide, an abasic nucleotide, a 2′-amino-modifiednucleotide, a 2′-O-allyl-modified nucleotide, 2′-C-alkyl-modifiednucleotide, 2′-hydroxyl-modified nucleotide, a 2′-methoxyethyl modifiednucleotide, a 2′-O-alkyl-modified nucleotide, a morpholino nucleotide, aphosphoramidate, a non-natural base comprising nucleotide, atetrahydropyran modified nucleotide, a 1,5-anhydrohexitol modifiednucleotide, a cyclohexenyl modified nucleotide, a nucleotide comprisinga phosphorothioate group, a nucleotide comprising a methylphosphonategroup, a nucleotide comprising a 5′-phosphate, and a nucleotidecomprising a 5′-phosphate mimic.

In one embodiment, the ligand is

In one embodiment, the RNAi agent is conjugated to the ligand as shownin the following schematic

wherein X is O or S.

In one embodiment, the subject is a human.

In one embodiment, the HDV-associated disorder is selected from thegroup consisting of hepatitis B virus infection, acute hepatitis B,acute hepatitis D; acute fulminant hepatitis D; chronic hepatitis D;liver fibrosis; end-stage liver disease; hepatocellular carcinoma.

In some embodiments, the methods of the invention further comprisetreatment of hepatitis B virus (HBV) in the subject. Methods oftreatment can include any methods of treatment known in the art. Incertain embodiments, HBV is treated in the subject using one of more ifthe iRNA agents provided herein.

In some embodiments, the methods of the invention further includemethods to modulate, preferably decrease the expression of PD-L1.Compositions and methods to reduce the expression of PD-L1 are provided,for example, in PCT publication no. WO2011/127180, which is herebyincorporated by reference.

In one embodiment, the double stranded RNAi agent is administered at adose of about 0.01 mg/kg to about 10 mg/kg or about 0.5 mg/kg to about50 mg/kg.

In one embodiment, the double stranded RNAi agent is administered at adose of about 10 mg/kg to about 30 mg/kg. In another embodiment, thedouble stranded RNAi agent is administered at a dose of about 3 mg/kg.In one embodiment, the double stranded RNAi agent is administered at adose of about 10 mg/kg.

In one embodiment, the double stranded RNAi agent is administered at adose of about 0.5 mg/kg twice per week.

In one embodiment, the double stranded RNAi agent is administered at afixed dose of about 50 mg to 200 mg.

In one embodiment, the double stranded RNAi agent is administeredsubcutaneously. In another embodiment, the double stranded RNAi agent isadministered intravenously. In another embodiment, the agent isadministered intramuscularly.

In one embodiment, the RNAi agent is administered in two or more doses.

In one embodiment, the RNAi agent is administered at intervals selectedfrom the group consisting of once every about 12 hours, once every about24 hours, once every about 48 hours, once every about 72 hours, and onceevery about 96 hours.

In one embodiment, the RNAi agent is administered twice per week. Inanother embodiment, the RNAi agent is administered every other week.

In one embodiment, the methods of the invention further includeadministering to the subject an additional therapeutic agent.

In one embodiment, the additional therapeutic agent is selected from thegroup consisting of an antiviral agent, a reverse transcriptaseinhibitor, an immune stimulator, a therapeutic vaccine, a viral entryinhibitor, an oligonucleotide that inhibits the secretion or release ofHbsAg, a capsid inhibitor, a cccDNA inhibitor, a double-stranded iRNAagent targeting HBV, and a combination of any of the foregoing.

In another embodiment, the methods of the invention further includeadministering administering to the subject a reverse transcriptaseinhibitor. In yet another embodiment, the methods of the inventionfurther include administering to the subject a reverse transcriptaseinhibitor and an immune stimulator.

In one embodiment, the reverse transcriptase inhibitor is selected fromthe group consisting of Tenofovir disoproxil fumarate (TDF), Tenofoviralafenamide, Lamivudine, Adefovir dipivoxil, Entecavir (ETV),Telbivudine, and AGX-1009.

In one embodiment, the immune stimulator is selected from the groupconsisting of pegylated interferon alfa 2a (PEG-IFN-α2a), Interferonalfa-2b, a recombinant human interleukin-7, and aToll-like receptor 7(TLR7) agonist.

In one embodiment, the double-stranded iRNA agent targeting HBV is anyone of the agents provided in any one of Tables 3, 4, 6, 14, 15, 24, 25,27, and 28. In another embodiment, the agent is AD-65403, AD-66810, or acombination thereof.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides iRNA compositions, which effect theRNA-induced silencing complex (RISC)-mediated cleavage of RNAtranscripts of a Hepatitis D virus (HDV) gene. The gene may be within acell, e.g., a cell within a subject, such as a human. The use of theseiRNAs enables the targeted degradation of mRNAs of the correspondinggene (HDV gene) in mammals.

The RNAi agents of the invention have been designed to target regions inthe HDV across various clades of HDV.

Also provided herein are HBV targeted RNAi agents of that have beendesigned to inhibit all steps of the HBV life cycle, e.g., replication,assembly, secretion of virus, and secretion of sub-viral antigens, byinhibiting expression of more than one HBV gene. In particular, sincetranscription of the HBV genome results in polycistronic, overlappingRNAs, an RNAi agent of the invention targeting a single HBV gene resultsin significant inhibition of expression of most or all HBV transcripts.For example, because the HBV genome is transcribed into a single mRNA,an RNAi agent of the invention targeting the S gene will result ininhibition of not only S gene expression but also the expression of the“downstream” reverse transcriptase gene. Furthermore, the RNAi agents ofthe invention have been designed to inhibit HBV viral replication bytargeting HBV structural genes, and the HBV X gene thereby permitting asubject's immune system to detect and respond to the presence of HBsAgto produce anti-HBV antibodies to clear an HBV infection.

HDV infection requires the presence of an HBV infection. Using in vitroassays, the present inventors have demonstrated that iRNA targeting HDVcan potently mediate RNAi, resulting in significant inhibition ofexpression of the HDV gene. Moreover, using in vitro and in vivo assays,it is demonstrated herein that iRNAs targeting an HBV gene can potentlymediate RNAi, resulting in significant inhibition of expression of morethan one HBV gene, is effective in treating HDV infection as well. Thus,methods and compositions including these iRNAs are useful for treating asubject having an HDV infection and/or an HDV-associated disease, byadministration of a iRNA targeted to one or both of HBV and HDV.Further, the compositions provided herein can be used in a subject withan HBV infection to prevent the development of an HDV infection.

Accordingly, the present invention also provides methods for treating asubject having a disorder that would benefit from inhibiting or reducingthe expression of an HBV gene, e.g., HDV infection, using iRNAcompositions which effect the RNA-induced silencing complex(RISC)-mediated cleavage of RNA transcripts of an HBV gene.

Very low dosages of the iRNAs of the invention, in particular, canspecifically and efficiently mediate RNA interference (RNAi), resultingin significant inhibition of expression of the corresponding gene (HDVgene).

The iRNAs of the invention include an RNA strand (the antisense strand)having a region which is about 30 nucleotides or less in length, e.g.,15-30, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21,15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25,18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26,19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27,20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27,21-26, 21-25, 21-24, 21-23, or 21-22 nucleotides in length, which regionis substantially complementary to at least part of an mRNA transcript ofan HBV gene.

The following detailed description discloses how to make and usecompositions containing iRNAs to inhibit the expression of anangiotensinogen gene as well as compositions, uses, and methods fortreating subjects having diseases and disorders that would benefit frominhibition and/or reduction of the expression of an HDV gene.

I. Definitions

In order that the present invention may be more readily understood,certain terms are first defined. In addition, it should be noted thatwhenever a value or range of values of a parameter are recited, it isintended that values and ranges intermediate to the recited values arealso intended to be part of this invention.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e., to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element, e.g., a plurality of elements.

The term “including” is used herein to mean, and is used interchangeablywith, the phrase “including but not limited to”.

The term “or” is used herein to mean, and is used interchangeably with,the term “and/or,” unless context clearly indicates otherwise.

As used herein, “Hepatitis D virus,” used interchangeably with the term“HDV” refers to the well-known noncytopathic, liver-tropic DNA virusbelonging to the Hepadnaviridae family. See, e.g., Ciancio and Rizzetto,Nat. Rev. 11:68-71, 2014; Le Gal et al., Emerg. Infect. Dis.12:1447-1450, 2006; and Abbas and afzal, World J. Hep., 5:666-675, 2013,all of which are incorporated by reference. Unless otherwise indicate,HDV refers to all clades and variants of HDV.

HDV produces one protein, namely HDAg. It comes in two forms; a 27 kDalarge-HDAg (also referred to herein as 1HD and large HDV antigen), and asmall-HDAg of 24 kDa (also referred to herein as sHD and small HDVantigen). The N-terminals of the two forms are identical, they differ by19 more amino acids in the C-terminal of the large HDAg. Both isoformsare produced from the same reading frame which contains an UAG stopcodon at codon 196, which normally produces only the small-HDAg.However, editing by cellular enzyme adenosine deaminase-1 changes thestop codon to UCG, allowing the large-HDAg to be produced. Despitehaving 90% identical sequences, these two proteins play diverging rolesduring the course of an infection. HDAg-S is produced in the earlystages of an infection and enters the nucleus and supports viralreplication. HDAg-L, in contrast, is produced during the later stages ofan infection, acts as an inhibitor of viral replication, and is requiredfor assembly of viral particles.

Additional examples of HDV mRNA sequences are readily available usingpublicly available databases, e.g., GenBank, UniProt, and OMIM.

The term “HDV,” as used herein, also refers to naturally occurring DNAsequence variations of the HDV genome.

As used herein, “Hepatitis B virus,” used interchangeably with the term“HBV” refers to the well-known noncytopathic, liver-tropic DNA virusbelonging to the Hepadnaviridae family.

The HBV genome is partially double-stranded, circular DNA withoverlapping reading frames.

There are four known genes encoded by the HBC genome, called C, X, P,and S. The core protein is coded for by gene C (HBcAg). Hepatitis Bantigen (HBeAg) is produced by proteolytic processing of the pre-core(pre-C) protein. The DNA polymerase is encoded by gene P. Gene S is thegene that codes for the surface antigen (HBsAg). The HBsAg gene is onelong open reading frame but contains three in frame “start” (ATG) codonsthat divide the gene into three sections, pre-S1, pre-S2, and S. Becauseof the multiple start codons, polypeptides of three different sizescalled large, middle, and small (pre-S1+pre-S2+S, pre-S2+S, or S) areproduced. The function of the non-structural protein coded for by gene Xis not fully understood but it is associated with the development ofliver cancer and encodes a decoy protein which permits HBsAg in theblood to sequester anti-HBsAg antibodies and allow infectious viralparticles to escape immune detection.

The proteins encoded by the HBV genome include: envelope proteins—i)small, Hepatitis B surface antigen (HBsAg); ii) middle—preS2 plus HBsAg;iii) large—preS1 plus preS2 plus HBsAg; nucleocapsid protein, hepatitisB core antigen (HBcAg). Hepatitis B e antigen (HBeAg) is anon-structural protein produced during the HBV replication which shares90% amino acids with the nucleocapsid HBcAg; and the X protein is anonstructural protein (HBx) which functions in the cytoplasm to activatevarious signaling pathways, many of which are controlled by modulationof cytosolic calcium and in the nucleus to regulate transcriptionthrough a direct interaction with different transcription factors and,in some cases, enhance their binding to specific transcription elements.

HBV is one of the few DNA viruses that utilize reverse transcriptase inthe replication process which involves multiple stages including entry,uncoating and transport of the virus genome to the nucleus. Initially,replication of the HBV genome involves the generation of an RNAintermediate that is then reverse transcribed to produce the DNA viralgenome.

Upon infection of a cell with HBV, the viral genomic relaxed circularDNA (rcDNA) is transported into the cell nucleus and converted intoepisomal covalently closed circular DNA (cccDNA), which serves as thetranscription template for the viral mRNAs. After transcription andnuclear export, cytoplasmic viral pregenomic RNA (pgRNA) is assembledwith HBV polymerase and capsid proteins to form the nucleocapsid, insidewhich polymerase-catalyzed reverse transcription yields minus-strandDNA, which is subsequently copied into plus-strand DNA to form theprogeny rcDNA genome. The mature nucleocapsids are then either packagedwith viral envelope proteins to egress as virion particles or shuttledto the nucleus to amplify the cccDNA reservoir through the intracellularcccDNA amplification pathway. cccDNA is an essential component of theHBV replication cycle and is responsible for the establishment ofinfection and viral persistence.

HBV infection results in the production of two different particles: 1)the HBV virus itself (or Dane particle) which includes a viral capsidassembled from the HBcAg and is covered by the HBsAg and is capable ofreinfecting cells and 2) subviral particles (or SVPs) which are highdensity lipoprotein-like particles comprised of lipids, cholesterol,cholesterol esters and the small and medium forms of the hepatitis Bsurface antigen HBsAg which are non-infectious. For each viral particleproduced, 1,000-10,000 SVPs are released into the blood. As such SVPs(and the HBsAg protein they carry) represent the overwhelming majorityof viral protein in the blood. HBV infected cells also secrete a solubleproteolytic product of the pre-core protein called the HBV e-antigen(HBeAg).

Eight genotypes of HBV, designated A to H, have been determined, eachhaving a distinct geographical distribution. The virus isnon-cytopathic, with virus-specific cellular immunity being the maindeterminant for the outcome of exposure to HBV-acute infection withresolution of liver diseases with 6 months, or chronic HBV infectionthat is frequently associated with progressive liver injury.

The term “HBV” includes any of the eight genotypes of HBV (A to H). Theamino acid and complete coding sequence of the reference sequence of theHBV genome may be found in for example, GenBank Accession Nos.GI:21326584 (SEQ ID NO:1) and GI:3582357 (SEQ ID NO:3).

Additional examples of HBV mRNA sequences are readily available usingpublicly available databases, e.g., GenBank, UniProt, and OMIM.

The term “HBV,” as used herein, also refers to naturally occurring DNAsequence variations of the HBV genome.

As used herein, “target sequence” refers to a contiguous portion of thenucleotide sequence of an mRNA molecule formed during the transcriptionof an HDV gene, including mRNA that is a product of RNA processing of aprimary transcription product. In one embodiment, the target portion ofthe sequence will be at least long enough to serve as a substrate foriRNA-directed cleavage at or near that portion of the nucleotidesequence of an mRNA molecule formed during the transcription of an HDVgene.

The target sequence may be from about 9-36 nucleotides in length, e.g.,about 15-30 nucleotides in length. For example, the target sequence canbe from about 15-30 nucleotides, 15-29, 15-28, 15-27, 15-26, 15-25,15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29,18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30,19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20,20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21,21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22nucleotides in length. Ranges and lengths intermediate to the aboverecited ranges and lengths are also contemplated to be part of theinvention.

As used herein, the term “strand comprising a sequence” refers to anoligonucleotide comprising a chain of nucleotides that is described bythe sequence referred to using the standard nucleotide nomenclature.

“G,” “C,” “A,” “T” and “U” each generally stand for a nucleotide thatcontains guanine, cytosine, adenine, thymidine and uracil as a base,respectively. However, it will be understood that the term“ribonucleotide” or “nucleotide” can also refer to a modifiednucleotide, as further detailed below, or a surrogate replacement moiety(see, e.g., Table 2). The skilled person is well aware that guanine,cytosine, adenine, and uracil can be replaced by other moieties withoutsubstantially altering the base pairing properties of an oligonucleotidecomprising a nucleotide bearing such replacement moiety. For example,without limitation, a nucleotide comprising inosine as its base can basepair with nucleotides containing adenine, cytosine, or uracil. Hence,nucleotides containing uracil, guanine, or adenine can be replaced inthe nucleotide sequences of dsRNA featured in the invention by anucleotide containing, for example, inosine. In another example, adenineand cytosine anywhere in the oligonucleotide can be replaced withguanine and uracil, respectively to form G-U Wobble base pairing withthe target mRNA. Sequences containing such replacement moieties aresuitable for the compositions and methods featured in the invention.

The terms “iRNA”, “RNAi agent,” “iRNA agent,”, “RNA interference agent”as used interchangeably herein, refer to an agent that contains RNA asthat term is defined herein, and which mediates the targeted cleavage ofan RNA transcript via an RNA-induced silencing complex (RISC) pathway.iRNA directs the sequence-specific degradation of mRNA through a processknown as RNA interference (RNAi). The iRNA modulates, e.g., inhibits,the expression of an HDV gene (e.g., one or more HDV genes) in a cell,e.g., a cell within a subject, such as a mammalian subject.

In one embodiment, an RNAi agent of the invention includes a singlestranded RNA that interacts with a target RNA sequence, e.g., an HDVtarget mRNA sequence, to direct the cleavage of the target RNA. Withoutwishing to be bound by theory it is believed that long double strandedRNA introduced into cells is broken down into siRNA by a Type IIIendonuclease known as Dicer (Sharp et al. (2001) Genes Dev. 15:485).Dicer, a ribonuclease-III-like enzyme, processes the dsRNA into 19-23base pair short interfering RNAs with characteristic two base 3′overhangs (Bernstein, et al., (2001) Nature 409:363). The siRNAs arethen incorporated into an RNA-induced silencing complex (RISC) where oneor more helicases unwind the siRNA duplex, enabling the complementaryantisense strand to guide target recognition (Nykanen, et al., (2001)Cell 107:309). Upon binding to the appropriate target mRNA, one or moreendonucleases within the RISC cleave the target to induce silencing(Elbashir, et al., (2001) Genes Dev. 15:188). Thus, in one aspect theinvention relates to a single stranded RNA (siRNA) generated within acell and which promotes the formation of a RISC complex to effectsilencing of the target gene, i.e., an HDV gene. Accordingly, the term“siRNA” is also used herein to refer to an RNAi as described above.

In another embodiment, the RNAi agent may be a single-stranded siRNAthat is introduced into a cell or organism to inhibit a target mRNA.Single-stranded RNAi agents bind to the RISC endonuclease, Argonaute 2,which then cleaves the target mRNA. The single-stranded siRNAs aregenerally 15-30 nucleotides and are chemically modified. The design andtesting of single-stranded siRNAs are described in U.S. Pat. No.8,101,348 and in Lima et al., (2012) Cell 150:883-894, the entirecontents of each of which are hereby incorporated herein by reference.Any of the antisense nucleotide sequences described herein may be usedas a single-stranded siRNA as described herein or as chemically modifiedby the methods described in Lima et al., (2012) Cell 150:883-894.

In another embodiment, an “iRNA” for use in the compositions, uses, andmethods of the invention is a double-stranded RNA and is referred toherein as a “double stranded RNAi agent,” “double-stranded RNA (dsRNA)molecule,” “dsRNA agent,” or “dsRNA”. The term “dsRNA”, refers to acomplex of ribonucleic acid molecules, having a duplex structurecomprising two anti-parallel and substantially complementary nucleicacid strands, referred to as having “sense” and “antisense” orientationswith respect to a target RNA, i.e., an HDV gene. In some embodiments ofthe invention, a double-stranded RNA (dsRNA) triggers the degradation ofa target RNA, e.g., an mRNA, through a post-transcriptionalgene-silencing mechanism referred to herein as RNA interference or RNAi.

In general, the majority of nucleotides of each strand of a dsRNAmolecule are ribonucleotides, but as described in detail herein, each orboth strands can also include one or more non-ribonucleotides, e.g., adeoxyribonucleotide and/or a modified nucleotide. In addition, as usedin this specification, an “RNAi agent” may include ribonucleotides withchemical modifications; an RNAi agent may include substantialmodifications at multiple nucleotides. As used herein, the term“modified nucleotide” refers to a nucleotide having, independently, amodified sugar moiety, a modified internucleotide linkage, and/ormodified nucleobase. Thus, the term modified nucleotide encompassessubstitutions, additions or removal of, e.g., a functional group oratom, to internucleoside linkages, sugar moieties, or nucleobases. Themodifications suitable for use in the agents of the invention includeall types of modifications disclosed herein or known in the art. Anysuch modifications, as used in a siRNA type molecule, are encompassed by“RNAi agent” for the purposes of this specification and claims.

The duplex region may be of any length that permits specific degradationof a desired target RNA through a RISC pathway, and may range from about9 to 36 base pairs in length, e.g., about 15-30 base pairs in length,for example, about 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 base pairsin length, such as about 15-30, 15-29, 15-28, 15-27, 15-26, 15-25,15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29,18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30,19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20,20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21,21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 basepairs in length. Ranges and lengths intermediate to the above recitedranges and lengths are also contemplated to be part of the invention.

The two strands forming the duplex structure may be different portionsof one larger RNA molecule, or they may be separate RNA molecules. Wherethe two strands are part of one larger molecule, and therefore areconnected by an uninterrupted chain of nucleotides between the 3′-end ofone strand and the 5′-end of the respective other strand forming theduplex structure, the connecting RNA chain is referred to as a “hairpinloop.” A hairpin loop can comprise at least one unpaired nucleotide. Insome embodiments, the hairpin loop can comprise at least 2, at least 3,at least 4, at least 5, at least 6, at least 7, at least 8, at least 9,at least 10, at least 20, at least 23 or more unpaired nucleotides.

Where the two substantially complementary strands of a dsRNA arecomprised by separate RNA molecules, those molecules need not, but canbe covalently connected. Where the two strands are connected covalentlyby means other than an uninterrupted chain of nucleotides between the3′-end of one strand and the 5′-end of the respective other strandforming the duplex structure, the connecting structure is referred to asa “linker.” The RNA strands may have the same or a different number ofnucleotides. The maximum number of base pairs is the number ofnucleotides in the shortest strand of the dsRNA minus any overhangs thatare present in the duplex. In addition to the duplex structure, an RNAimay comprise one or more nucleotide overhangs.

In one embodiment, an RNAi agent of the invention is a dsRNA of 24-30nucleotides that interacts with a target RNA sequence, e.g., an HDVtarget mRNA sequence, to direct the cleavage of the target RNA. Withoutwishing to be bound by theory, long double stranded RNA introduced intocells is broken down into siRNA by a Type III endonuclease known asDicer (Sharp et al. (2001) Genes Dev. 15:485). Dicer, aribonuclease-III-like enzyme, processes the dsRNA into 19-23 base pairshort interfering RNAs with characteristic two base 3′ overhangs(Bernstein, et al., (2001) Nature 409:363). The siRNAs are thenincorporated into an RNA-induced silencing complex (RISC) where one ormore helicases unwind the siRNA duplex, enabling the complementaryantisense strand to guide target recognition (Nykanen, et al., (2001)Cell 107:309). Upon binding to the appropriate target mRNA, one or moreendonucleases within the RISC cleave the target to induce silencing(Elbashir, et al., (2001) Genes Dev. 15:188).

As used herein, the term “nucleotide overhang” refers to at least oneunpaired nucleotide that protrudes from the duplex structure of an iRNA,e.g., a dsRNA. For example, when a 3′-end of one strand of a dsRNAextends beyond the 5′-end of the other strand, or vice versa, there is anucleotide overhang. A dsRNA can comprise an overhang of at least onenucleotide; alternatively the overhang can comprise at least twonucleotides, at least three nucleotides, at least four nucleotides, atleast five nucleotides or more. A nucleotide overhang can comprise orconsist of a nucleotide/nucleoside analog, including adeoxynucleotide/nucleoside. The overhang(s) can be on the sense strand,the antisense strand or any combination thereof. Furthermore, thenucleotide(s) of an overhang can be present on the 5′-end, 3′-end orboth ends of either an antisense or sense strand of a dsRNA.

In one embodiment, the antisense strand of a dsRNA has a 1-10nucleotide, e.g., a 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide,overhang at the 3′-end and/or the 5′-end. In one embodiment, the sensestrand of a dsRNA has a 1-10 nucleotide, e.g., a 1, 2, 3, 4, 5, 6, 7, 8,9, or 10 nucleotide, overhang at the 3′-end and/or the 5′-end. Inanother embodiment, one or more of the nucleotides in the overhang isreplaced with a nucleoside thiophosphate.

“Blunt” or “blunt end” means that there are no unpaired nucleotides atthat end of the double stranded RNAi agent, i.e., no nucleotideoverhang. A “blunt ended” RNAi agent is a dsRNA that is double-strandedover its entire length, i.e., no nucleotide overhang at either end ofthe molecule. The RNAi agents of the invention include RNAi agents withnucleotide overhangs at one end (i.e., agents with one overhang and oneblunt end) or with nucleotide overhangs at both ends.

The term “antisense strand” or “guide strand” refers to the strand of aniRNA, e.g., a dsRNA, which includes a region that is substantiallycomplementary to a target sequence, e.g., a HDV mRNA. As used herein,the term “region of complementarity” refers to the region on theantisense strand that is substantially complementary to a sequence, forexample a target sequence, e.g., an HDV nucleotide sequence, as definedherein. Where the region of complementarity is not fully complementaryto the target sequence, the mismatches can be in the internal orterminal regions of the molecule. Generally, the most toleratedmismatches are in the terminal regions, e.g., within 5, 4, 3, 2, or 1nucleotides of the 5′- and/or 3′-terminus of the iRNA. In oneembodiment, a double-stranded RNAi agent of the invention include a anucleotide mismatch in the antisense strand. In another embodiment, adouble-stranded RNAi agent of the invention include a a nucleotidemismatch in the sense strand. In one embodiment, the nucleotide mismatchis, for example, within 5, 4, 3, 2, or 1 nucleotides from the3′-terminus of the iRNA. In another embodiment, the nucleotide mismatchis, for example, in the 3′-terminal nucleotide of the iRNA.

The term “sense strand,” or “passenger strand” as used herein, refers tothe strand of an iRNA that includes a region that is substantiallycomplementary to a region of the antisense strand as that term isdefined herein.

As used herein, the term “cleavage region” refers to a region that islocated immediately adjacent to the cleavage site. The cleavage site isthe site on the target at which cleavage occurs. In some embodiments,the cleavage region comprises three bases on either end of, andimmediately adjacent to, the cleavage site. In some embodiments, thecleavage region comprises two bases on either end of, and immediatelyadjacent to, the cleavage site. In some embodiments, the cleavage sitespecifically occurs at the site bound by nucleotides 10 and 11 of theantisense strand, and the cleavage region comprises nucleotides 11, 12and 13.

As used herein, and unless otherwise indicated, the term“complementary,” when used to describe a first nucleotide sequence inrelation to a second nucleotide sequence, refers to the ability of anoligonucleotide or polynucleotide comprising the first nucleotidesequence to hybridize and form a duplex structure under certainconditions with an oligonucleotide or polynucleotide comprising thesecond nucleotide sequence, as will be understood by the skilled person.Such conditions can, for example, be stringent conditions, wherestringent conditions can include: 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mMEDTA, 50° C. or 70° C. for 12-16 hours followed by washing (see, e.g.,“Molecular Cloning: A Laboratory Manual, Sambrook, et al. (1989) ColdSpring Harbor Laboratory Press). Other conditions, such asphysiologically relevant conditions as can be encountered inside anorganism, can apply. The skilled person will be able to determine theset of conditions most appropriate for a test of complementarity of twosequences in accordance with the ultimate application of the hybridizednucleotides.

Complementary sequences within an iRNA, e.g., within a dsRNA asdescribed herein, include base-pairing of the oligonucleotide orpolynucleotide comprising a first nucleotide sequence to anoligonucleotide or polynucleotide comprising a second nucleotidesequence over the entire length of one or both nucleotide sequences.Such sequences can be referred to as “fully complementary” with respectto each other herein. However, where a first sequence is referred to as“substantially complementary” with respect to a second sequence herein,the two sequences can be fully complementary, or they can form one ormore, but generally not more than 5, 4, 3 or 2 mismatched base pairsupon hybridization for a duplex up to 30 base pairs, while retaining theability to hybridize under the conditions most relevant to theirultimate application, e.g., inhibition of gene expression via a RISCpathway. However, where two oligonucleotides are designed to form, uponhybridization, one or more single stranded overhangs, such overhangsshall not be regarded as mismatches with regard to the determination ofcomplementarity. For example, a dsRNA comprising one oligonucleotide 21nucleotides in length and another oligonucleotide 23 nucleotides inlength, wherein the longer oligonucleotide comprises a sequence of 21nucleotides that is fully complementary to the shorter oligonucleotide,can yet be referred to as “fully complementary” for the purposesdescribed herein.

“Complementary” sequences, as used herein, can also include, or beformed entirely from, non-Watson-Crick base pairs and/or base pairsformed from non-natural and modified nucleotides, in so far as the aboverequirements with respect to their ability to hybridize are fulfilled.Such non-Watson-Crick base pairs include, but are not limited to, G:UWobble or Hoogstein base pairing.

The terms “complementary,” “fully complementary” and “substantiallycomplementary” herein can be used with respect to the base matchingbetween the sense strand and the antisense strand of a dsRNA, or betweenthe antisense strand of an iRNA agent and a target sequence, as will beunderstood from the context of their use.

As used herein, a polynucleotide that is “substantially complementary toat least part of” a messenger RNA (mRNA) refers to a polynucleotide thatis substantially complementary to a contiguous portion of the mRNA ofinterest (e.g., an mRNA encoding an HDV gene). For example, apolynucleotide is complementary to at least a part of an HDV mRNA if thesequence is substantially complementary to a non-interrupted portion ofan mRNA encoding an HDV gene.

Accordingly, in some embodiments, the sense strand polynucleotides andthe antisense polynucleotides disclosed herein are fully complementaryto the target HDV sequence. In other embodiments, the sense strandpolynucleotides and/or the antisense polynucleotides disclosed hereinare substantially complementary to the target HDV sequence and comprisea contiguous nucleotide sequence which is at least about 80%complementary over its entire length to the equivalent region of thenucleotide sequence of any one of the sequences in Tables 11, 12, 31,and 32, or a fragment of any one of the sequences in Tables 11, 12, 31,and 32, such as about 85%, about 86%, about 87%, about 88%, about 89%,about 90%, about % 91%, about 92%, about 93%, about 94%, about 95%,about 96%, about 97%, about 98%, or about 99% complementary.

In one embodiment, an RNAi agent of the invention includes a sensestrand that is substantially complementary to the target HDV sequenceand comprise a contiguous nucleotide sequence which is at least about80% complementary over its entire length to the equivalent region of thenucleotide sequence of any one of the sequences in Tables 11, 12, 31,and 32, or a fragment of any one of the sequences in Tables 11, 12, 31,and 32, such as about 85%, about 86%, about 87%, about 88%, about 89%,about 90%, about % 91%, about 92%, about 93%, about 94%, about 95%,about 96%, about 97%, about 98%, or about 99% complementary.

In another embodiment, an RNAi agent of the invention includes anantisense strand that is substantially complementary to the target HDVsequence and comprise a contiguous nucleotide sequence which is at leastabout 80% complementary over its entire length to the equivalent regionof the nucleotide sequence of any one of the sequences in Tables 11, 12,31, and 32, or a fragment of any one of the sequences in Tables 11, 12,31, and 32, such as about 85%, about 86%, about 87%, about 88%, about89%, about 90%, about % 91%, about 92%, about 93%, about 94%, about 95%,about 96%, about 97%, about 98%, or about 99% complementary.

In some embodiments, the RNAi agents for use in the invention targetHBV. In some embodiment, such agents are fully complementary to thetarget HBV sequence. In other embodiments, the sense strandpolynucleotides and/or the antisense polynucleotides disclosed hereinare substantially complementary to the target HBV sequence and comprisea contiguous nucleotide sequence which is at least about 80%complementary over its entire length to the equivalent region of thenucleotide sequence of any one of the nucleotide sequences of the agentstargeting HBV provided herein, such as about 85%, about 86%, about 87%,about 88%, about 89%, about 90%, about % 91%, about 92%, about 93%,about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%complementary.

In one embodiment, an RNAi agent targeting HBV for use in the presentinvention includes a sense strand that is substantially complementary tothe target HBV sequence and comprise a contiguous nucleotide sequencewhich is at least about 80% complementary over its entire length to theequivalent region of the nucleotide sequence of any one of thenucleotide sequences of the agents targeting HBV provided herein, suchas about 85%, about 86%, about 87%, about 88%, about 89%, about 90%,about % 91%, about 92%, about 93%, about 94%, about 95%, about 96%,about 97%, about 98%, or about 99% complementary.

In another embodiment, an RNAi agent targeting HBV for use in thepresent invention includes an antisense strand that is substantiallycomplementary to the target HBV sequence and comprise a contiguousnucleotide sequence which is at least about 80% complementary over itsentire length to the equivalent region of the nucleotide sequence of anyone of the nucleotide sequences of the agents targeting HBV providedherein, or a fragment of any one of the nucleotide sequences of theagents targeting HBV provided herein, such as about 85%, about 86%,about 87%, about 88%, about 89%, about 90%, about % 91%, about 92%,about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, orabout 99% complementary.

In general, the majority of nucleotides of each strand areribonucleotides, but as described in detail herein, each or both strandscan also include one or more non-ribonucleotides, e.g., adeoxyribonucleotide and/or a modified nucleotide. In addition, an “iRNA”may include ribonucleotides with chemical modifications. Suchmodifications may include all types of modifications disclosed herein orknown in the art. Any such modifications, as used in an iRNA molecule,are encompassed by “iRNA” for the purposes of this specification andclaims.

In one aspect of the invention, an agent for use in the methods andcompositions of the invention is a single-stranded antisense RNAmolecule that inhibits a target mRNA via an antisense inhibitionmechanism. The single-stranded antisense RNA molecule is complementaryto a sequence within the target mRNA. The single-stranded antisenseoligonucleotides can inhibit translation in a stoichiometric manner bybase pairing to the mRNA and physically obstructing the translationmachinery, see Dias, N. et al., (2002) Mol Cancer Ther 1:347-355. Thesingle-stranded antisense RNA molecule may be about 15 to about 30nucleotides in length and have a sequence that is complementary to atarget sequence. For example, the single-stranded antisense RNA moleculemay comprise a sequence that is at least about 15, 16, 17, 18, 19, 20,or more contiguous nucleotides from any one of the antisense sequencesdescribed herein.

As used herein, a “subject” is an animal, such as a mammal, including aprimate (such as a human, a non-human primate, e.g., a monkey, and achimpanzee), a non-primate (such as a cow, a pig, a camel, a llama, ahorse, a goat, a rabbit, a sheep, a hamster, a guinea pig, a cat, a dog,a rat, a mouse, a horse, and a whale), or a bird (e.g., a duck or agoose). In an embodiment, the subject is a human, such as a human beingtreated or assessed for a disease, disorder or condition that wouldbenefit from reduction in HDV gene expression and/or replication; ahuman at risk for a disease, disorder or condition that would benefitfrom reduction in HDV gene expression and/or replication; a human havinga disease, disorder or condition that would benefit from reduction inHDV gene expression and/or replication; and/or human being treated for adisease, disorder or condition that would benefit from reduction in HDVgene expression and/or replication, as described herein.

As used herein, the terms “treating” or “treatment” refer to abeneficial or desired result including, but not limited to, alleviationor amelioration of one or more symptoms associated with unwanted HDVgene expression and/or HDV replication. “Treatment” can also meanprolonging survival as compared to expected survival in the absence oftreatment.

The term “lower” in the context of the level of HDV gene expressionand/or HDV replication in a subject or a disease marker or symptomrefers to a statistically significant decrease in such level. Thedecrease can be, for example, at least 10%, at least 15%, at least 20%,at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, atleast 50%, at least 55%, at least 60%, at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, ormore and is preferably down to a level accepted as within the range ofnormal for an individual without such disorder. In certain embodiments,the expression of the target is normalized, i.e., decreased to a levelaccepted as within the range of normal for an individual without suchdisorder, e.g., the level of a disease marker, such as, ALT or AST, isdecreased to a level accepted as within the range of normal for anindividual without such disorder.

As used herein, “prevention” or “preventing,” when used in reference toa disease, disorder or condition thereof, that would benefit from areduction in expression of an HDV gene and/or replication, refers to areduction in the likelihood that a subject will develop a symptomassociated with such a disease, disorder, or condition, e.g., a symptomof unwanted HDV infection, such as the presence of serum and/or liverHDV ccc DNA, the presence of serum HDV DNA, the presence of serum and/orliver HDV antigen.

As used herein, the term “Hepatitis D virus-associated disease” or“HDV-associated disease,” is a disease or disorder that is caused by, orassociated with HDV infection and/or replication. The term“HDV-associated disease” includes a disease, disorder or condition thatwould benefit from reduction in HDV gene expression and/or replication.Non-limiting examples of HDV-associated diseases include, for example,hepatitis B virus infection, acute hepatitis B, acute hepatitis D; acutefulminant hepatitis D; chronic hepatitis D; liver fibrosis; end-stageliver disease; hepatocellular carcinoma.

“Therapeutically effective amount,” as used herein, is intended toinclude the amount of an RNAi agent that, when administered to a patientfor treating a subject having an HDV infection and/or HDV-associateddisease, is sufficient to effect treatment of the disease (e.g., bydiminishing, ameliorating or maintaining the existing disease or one ormore symptoms of disease). The “therapeutically effective amount” mayvary depending on the RNAi agent, how the agent is administered, thedisease and its severity and the history, age, weight, family history,genetic makeup, stage of pathological processes mediated by HDV geneexpression, the types of preceding or concomitant treatments, if any,and other individual characteristics of the patient to be treated.

“Prophylactically effective amount,” as used herein, is intended toinclude the amount of an RNAi agent that, when administered to a subjectwho does not yet experience or display symptoms of an HDV infectionand/or HDV-associated disease, but who may be predisposed, is sufficientto prevent or ameliorate the disease or one or more symptoms of thedisease. Ameliorating the disease includes slowing the course of thedisease or reducing the severity of later-developing disease. The“prophylactically effective amount” may vary depending on the RNAiagent, how the agent is administered, the degree of risk of disease, andthe history, age, weight, family history, genetic makeup, the types ofpreceding or concomitant treatments, if any, and other individualcharacteristics of the patient to be treated.

A “therapeutically-effective amount” or “prophylactically effectiveamount” also includes an amount of an RNAi agent that produces somedesired local or systemic effect at a reasonable benefit/risk ratioapplicable to any treatment. RNAi agents employed in the methods of thepresent invention may be administered in a sufficient amount to producea reasonable benefit/risk ratio applicable to such treatment.

The term “sample,” as used herein, includes a collection of similarfluids, cells, or tissues isolated from a subject, as well as fluids,cells, or tissues present within a subject. Examples of biologicalfluids include blood, serum and serosal fluids, plasma, cerebrospinalfluid, ocular fluids, lymph, urine, saliva, and the like. Tissue samplesmay include samples from tissues, organs or localized regions. Forexample, samples may be derived from particular organs, parts of organs,or fluids or cells within those organs. In certain embodiments, samplesmay be derived from the liver (e.g., whole liver or certain segments ofliver or certain types of cells in the liver, such as, e.g.,hepatocytes), the retina or parts of the retina (e.g., retinal pigmentepithelium), the central nervous system or parts of the central nervoussystem (e.g., ventricles or choroid plexus), or the pancreas or certaincells or parts of the pancreas. In some embodiments, a “sample derivedfrom a subject” refers tocerebrospinal fluid obtained from the subject.In preferred embodiments, a “sample derived from a subject” refers toblood or plasma drawn from the subject. In further embodiments, a“sample derived from a subject” refers to liver tissue (or subcomponentsthereof) or retinal tissue (or subcomponents thereof) derived from thesubject.

II. iRNAs of the Invention

The present invention provides iRNAs which inhibit the expression of oneor more HDV genes. In one embodiment, the iRNA agent includesdouble-stranded ribonucleic acid (dsRNA) molecules for inhibiting theexpression of an HDV gene in a cell, such as a cell within a subject,e.g., a mammal, such as a human having an HDV-associated disease, e.g.,chronic hepatitis D. The dsRNA includes an antisense strand having aregion of complementarity which is complementary to at least a part ofan mRNA formed in the expression of an HDV gene. The region ofcomplementarity is about 30 nucleotides or less in length (e.g., about30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, or 18 nucleotides orless in length). Upon contact with a cell expressing the HDV gene, theiRNA inhibits the expression of the HDV gene (e.g., a human, a primate,a non-primate, or a bird HDV gene) by at least about 10% as assayed by,for example, a PCR or branched DNA (bDNA)-based method, or by aprotein-based method, such as by immunofluorescence analysis, using, forexample, western Blotting or flowcytometric techniques.

A dsRNA includes two RNA strands that are complementary and hybridize toform a duplex structure under conditions in which the dsRNA will beused. One strand of a dsRNA (the antisense strand) includes a region ofcomplementarity that is substantially complementary, and generally fullycomplementary, to a target sequence. The target sequence can be derivedfrom the sequence of an mRNA formed during the expression of an HDVgene. The other strand (the sense strand) includes a region that iscomplementary to the antisense strand, such that the two strandshybridize and form a duplex structure when combined under suitableconditions. As described elsewhere herein and as known in the art, thecomplementary sequences of a dsRNA can also be contained asself-complementary regions of a single nucleic acid molecule, as opposedto being on separate oligonucleotides.

Generally, the duplex structure is between 15 and 30 base pairs inlength, e.g., between, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23,15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27,18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28,19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29,20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29,21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 base pairs in length.Ranges and lengths intermediate to the above recited ranges and lengthsare also contemplated to be part of the invention.

Similarly, the region of complementarity to the target sequence isbetween 15 and 30 nucleotides in length, e.g., between 15-29, 15-28,15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18,15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22,18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23,19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24,20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24,21-23, or 21-22 nucleotides in length. Ranges and lengths intermediateto the above recited ranges and lengths are also contemplated to be partof the invention.

In some embodiments, the dsRNA is between about 15 and about 20nucleotides in length, or between about 25 and about 30 nucleotides inlength. In general, the dsRNA is long enough to serve as a substrate forthe Dicer enzyme. For example, it is well-known in the art that dsRNAslonger than about 21-23 nucleotides in length may serve as substratesfor Dicer. As the ordinarily skilled person will also recognize, theregion of an RNA targeted for cleavage will most often be part of alarger RNA molecule, often an mRNA molecule. Where relevant, a “part” ofan mRNA target is a contiguous sequence of an mRNA target of sufficientlength to allow it to be a substrate for RNAi-directed cleavage (i.e.,cleavage through a RISC pathway).

One of skill in the art will also recognize that the duplex region is aprimary functional portion of a dsRNA, e.g., a duplex region of about 9to 36 base pairs, e.g., about 10-36, 11-36, 12-36, 13-36, 14-36, 15-36,9-35, 10-35, 11-35, 12-35, 13-35, 14-35, 15-35, 9-34, 10-34, 11-34,12-34, 13-34, 14-34, 15-34, 9-33, 10-33, 11-33, 12-33, 13-33, 14-33,15-33, 9-32, 10-32, 11-32, 12-32, 13-32, 14-32, 15-32, 9-31, 10-31,11-31, 12-31, 13-32, 14-31, 15-31, 15-30, 15-29, 15-28, 15-27, 15-26,15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30,18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20,19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21,19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22,20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22base pairs. Thus, in one embodiment, to the extent that it becomesprocessed to a functional duplex, of e.g., 15-30 base pairs, thattargets a desired RNA for cleavage, an RNA molecule or complex of RNAmolecules having a duplex region greater than 30 base pairs is a dsRNA.Thus, an ordinarily skilled artisan will recognize that in oneembodiment, a miRNA is a dsRNA. In another embodiment, a dsRNA is not anaturally occurring miRNA. In another embodiment, an iRNA agent usefulto target HDV gene expression is not generated in the target cell bycleavage of a larger dsRNA.

A dsRNA as described herein can further include one or moresingle-stranded nucleotide overhangs e.g., 1, 2, 3, or 4 nucleotides.dsRNAs having at least one nucleotide overhang can have unexpectedlysuperior inhibitory properties relative to their blunt-endedcounterparts. A nucleotide overhang can comprise or consist of anucleotide/nucleoside analog, including a deoxynucleotide/nucleoside.The overhang(s) can be on the sense strand, the antisense strand or anycombination thereof. Furthermore, the nucleotide(s) of an overhang canbe present on the 5′-end, 3′-end or both ends of either an antisense orsense strand of a dsRNA.

A dsRNA can be synthesized by standard methods known in the art asfurther discussed below, e.g., by use of an automated DNA synthesizer,such as are commercially available from, for example, Biosearch, AppliedBiosystems, Inc.

iRNA compounds of the invention may be prepared using a two-stepprocedure. First, the individual strands of the double-stranded RNAmolecule are prepared separately. Then, the component strands areannealed. The individual strands of the siRNA compound can be preparedusing solution-phase or solid-phase organic synthesis or both. Organicsynthesis offers the advantage that the oligonucleotide strandscomprising unnatural or modified nucleotides can be easily prepared.Single-stranded oligonucleotides of the invention can be prepared usingsolution-phase or solid-phase organic synthesis or both.

In one aspect, a dsRNA of the invention includes at least two nucleotidesequences, a sense sequence and an anti-sense sequence. The sense strandis selected from the group of sequences provided in any one of Tables11, 12, 31, and 32 and the corresponding antisense strand of the sensestrand is selected from the group of sequences of any one of Tables 11,12, 31, and 32. In this aspect, one of the two sequences iscomplementary to the other of the two sequences, with one of thesequences being substantially complementary to a sequence of an mRNAgenerated in the expression of an HDV gene. As such, in this aspect, adsRNA will include two oligonucleotides, where one oligonucleotide isdescribed as the sense strand in any one of Tables 11, 12, 31, and 32and the second oligonucleotide is described as the correspondingantisense strand of the sense strand in any one of Tables 11, 12, 31,and 32. In one embodiment, the substantially complementary sequences ofthe dsRNA are contained on separate oligonucleotides. In anotherembodiment, the substantially complementary sequences of the dsRNA arecontained on a single oligonucleotide.

It will be understood that, although some of the sequences in Tables 12and 32 are described as modified and/or conjugated sequences, the RNA ofthe iRNA of the invention e.g., a dsRNA of the invention, may compriseany one of the sequences set forth in any one of Tables 11, 12, 31, and32 that is un-modified, un-conjugated, and/or modified and/or conjugateddifferently than described therein.

The skilled person is well aware that dsRNAs having a duplex structureof between about 20 and 23 base pairs, e.g., 21, base pairs have beenhailed as particularly effective in inducing RNA interference (Elbashiret al., EMBO 2001, 20:6877-6888). However, others have found thatshorter or longer RNA duplex structures can also be effective (Chu andRana (2007) RNA 14:1714-1719; Kim et al. (2005) Nat Biotech 23:222-226).In the embodiments described above, by virtue of the nature of theoligonucleotide sequences provided in any one of Tables 11, 12, 31, and32, dsRNAs described herein can include at least one strand of a lengthof minimally 21 nucleotides. It can be reasonably expected that shorterduplexes having one of the sequences of any one of Tables 11, 12, 31,and 32 minus only a few nucleotides on one or both ends can be similarlyeffective as compared to the dsRNAs described above. Hence, dsRNAshaving a sequence of at least 15, 16, 17, 18, 19, 20, or more contiguousnucleotides derived from one of the sequences of any one of Tables 11,12, 31, and 32, and differing in their ability to inhibit the expressionof a HDV gene by not more than about 5, 10, 15, 20, 25, or 30%inhibition from a dsRNA comprising the full sequence, are contemplatedto be within the scope of the present invention.

In addition, the RNAs provided in any one of Tables 11, 12, 31, and 32identify a site(s) in a HDV transcript that is susceptible toRISC-mediated cleavage. As such, the present invention further featuresiRNAs that target within one of these sites. As used herein, an iRNA issaid to target within a particular site of an RNA transcript if the iRNApromotes cleavage of the transcript anywhere within that particularsite. Such an iRNA will generally include at least about 15 contiguousnucleotides from one of the sequences provided inany one of Tables 11,12, 31, and 32 coupled to additional nucleotide sequences taken from theregion contiguous to the selected sequence in a HDV gene.

While a target sequence is generally about 15-30 nucleotides in length,there is wide variation in the suitability of particular sequences inthis range for directing cleavage of any given target RNA. Varioussoftware packages and the guidelines set out herein provide guidance forthe identification of optimal target sequences for any given genetarget, but an empirical approach can also be taken in which a “window”or “mask” of a given size (as a non-limiting example, 21 nucleotides) isliterally or figuratively (including, e.g., in silico) placed on thetarget RNA sequence to identify sequences in the size range that canserve as target sequences. By moving the sequence “window” progressivelyone nucleotide upstream or downstream of an initial target sequencelocation, the next potential target sequence can be identified, untilthe complete set of possible sequences is identified for any giventarget size selected. This process, coupled with systematic synthesisand testing of the identified sequences (using assays as describedherein or as known in the art) to identify those sequences that performoptimally can identify those RNA sequences that, when targeted with aniRNA agent, mediate the best inhibition of target gene expression. Thus,while the sequences identified, for example, in any one of Tables 11,12, 31, and 32 represent effective target sequences, it is contemplatedthat further optimization of inhibition efficiency can be achieved byprogressively “walking the window” one nucleotide upstream or downstreamof the given sequences to identify sequences with equal or betterinhibition characteristics.

Further, it is contemplated that for any sequence identified, e.g., inany one of Tables 11, 12, 31, and 32, further optimization could beachieved by systematically either adding or removing nucleotides togenerate longer or shorter sequences and testing those sequencesgenerated by walking a window of the longer or shorter size up or downthe target RNA from that point. Again, coupling this approach togenerating new candidate targets with testing for effectiveness of iRNAsbased on those target sequences in an inhibition assay as known in theart and/or as described herein can lead to further improvements in theefficiency of inhibition. Further still, such optimized sequences can beadjusted by, e.g., the introduction of modified nucleotides as describedherein or as known in the art, addition or changes in overhang, or othermodifications as known in the art and/or discussed herein to furtheroptimize the molecule (e.g., increasing serum stability or circulatinghalf-life, increasing thermal stability, enhancing transmembranedelivery, targeting to a particular location or cell type, increasinginteraction with silencing pathway enzymes, increasing release fromendosomes) as an expression inhibitor.

An iRNA as described herein can contain one or more mismatches to thetarget sequence. In one embodiment, an iRNA as described herein containsno more than 3 mismatches. If the antisense strand of the iRNA containsmismatches to a target sequence, it is preferable that the area ofmismatch is not located in the center of the region of complementarity.If the antisense strand of the iRNA contains mismatches to the targetsequence, it is preferable that the mismatch be restricted to be withinthe last 5 nucleotides from either the 5′- or 3′-end of the region ofcomplementarity. For example, for a 23 nucleotide iRNA agent the strandwhich is complementary to a region of an HDV gene, generally does notcontain any mismatch within the central 13 nucleotides. The methodsdescribed herein or methods known in the art can be used to determinewhether an iRNA containing a mismatch to a target sequence is effectivein inhibiting the expression of an HDV gene. Consideration of theefficacy of iRNAs with mismatches in inhibiting expression of an HDVgene is important, especially if the particular region ofcomplementarity in an HDV gene is known to have polymorphic sequencevariation within the population.

III. Modified iRNAs of the Invention

In one embodiment, the RNA of the iRNA of the invention e.g., a dsRNA,is un-modified, and does not comprise, e.g., chemical modificationsand/or conjugations known in the art and described herein. In anotherembodiment, the RNA of an iRNA of the invention, e.g., a dsRNA, ischemically modified to enhance stability or other beneficialcharacteristics. In certain embodiments of the invention, substantiallyall of the nucleotides of an iRNA of the invention are modified. Inother embodiments of the invention, all of the nucleotides of an iRNA ofthe invention are modified iRNAs of the invention in which“substantially all of the nucleotides are modified” are largely but notwholly modified and can include not more than 5, 4, 3, 2, or 1unmodified nucleotides.

The nucleic acids featured in the invention can be synthesized and/ormodified by methods well established in the art, such as those describedin “Current protocols in nucleic acid chemistry,” Beaucage, S. L. et al.(Edrs.), John Wiley & Sons, Inc., New York, N.Y., USA, which is herebyincorporated herein by reference. Modifications include, for example,end modifications, e.g., 5′-end modifications (phosphorylation,conjugation, inverted linkages) or 3′-end modifications (conjugation,DNA nucleotides, inverted linkages, etc.); base modifications, e.g.,replacement with stabilizing bases, destabilizing bases, or bases thatbase pair with an expanded repertoire of partners, removal of bases(abasic nucleotides), or conjugated bases; sugar modifications (e.g., atthe 2′-position or 4′-position) or replacement of the sugar; and/orbackbone modifications, including modification or replacement of thephosphodiester linkages. Specific examples of iRNA compounds useful inthe embodiments described herein include, but are not limited to RNAscontaining modified backbones or no natural internucleoside linkages.RNAs having modified backbones include, among others, those that do nothave a phosphorus atom in the backbone. For the purposes of thisspecification, and as sometimes referenced in the art, modified RNAsthat do not have a phosphorus atom in their internucleoside backbone canalso be considered to be oligonucleosides. In some embodiments, amodified iRNA will have a phosphorus atom in its internucleosidebackbone.

Modified RNA backbones include, for example, phosphorothioates, chiralphosphorothioates, phosphorodithioates, phosphotriesters,aminoalkylphosphotriesters, methyl and other alkyl phosphonatesincluding 3′-alkylene phosphonates and chiral phosphonates,phosphinates, phosphoramidates including 3′-amino phosphoramidate andaminoalkylphosphoramidates, thionophosphoramidates,thionoalkylphosphonates, thionoalkylphosphotriesters, andboranophosphates having normal 3′-5′ linkages, 2′-5′-linked analogs ofthese, and those having inverted polarity wherein the adjacent pairs ofnucleoside units are linked 3′-5′ to 5′-3′ or 2′-5′ to 5′-2′. Varioussalts, mixed salts and free acid forms are also included.

Representative U.S. patents that teach the preparation of the abovephosphorus-containing linkages include, but are not limited to, U.S.Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,195;5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131;5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925;5,519,126; 5,536,821; 5,541,316; 5,550,111; 5,563,253; 5,571,799;5,587,361; 5,625,050; 6,028,188; 6,124,445; 6,160,109; 6,169,170;6,172,209; 6,239,265; 6,277,603; 6,326,199; 6,346,614; 6,444,423;6,531,590; 6,534,639; 6,608,035; 6,683,167; 6,858,715; 6,867,294;6,878,805; 7,015,315; 7,041,816; 7,273,933; 7,321,029; and U.S. Pat.RE39464, the entire contents of each of which are hereby incorporatedherein by reference.

Modified RNA backbones that do not include a phosphorus atom thereinhave backbones that are formed by short chain alkyl or cycloalkylinternucleoside linkages, mixed heteroatoms and alkyl or cycloalkylinternucleoside linkages, or one or more short chain heteroatomic orheterocyclic internucleoside linkages. These include those havingmorpholino linkages (formed in part from the sugar portion of anucleoside); siloxane backbones; sulfide, sulfoxide and sulfonebackbones; formacetyl and thioformacetyl backbones; methylene formacetyland thioformacetyl backbones; alkene containing backbones; sulfamatebackbones; methyleneimino and methylenehydrazino backbones; sulfonateand sulfonamide backbones; amide backbones; and others having mixed N,O, S and CH₂ component parts.

Representative U.S. patents that teach the preparation of the aboveoligonucleosides include, but are not limited to, U.S. Pat. Nos.5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033;5,64,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967;5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,608,046;5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and,5,677,439, the entire contents of each of which are hereby incorporatedherein by reference.

In other embodiments, suitable RNA mimetics are contemplated for use iniRNAs, in which both the sugar and the internucleoside linkage, i.e.,the backbone, of the nucleotide units are replaced with novel groups.The base units are maintained for hybridization with an appropriatenucleic acid target compound. One such oligomeric compound, an RNAmimetic that has been shown to have excellent hybridization properties,is referred to as a peptide nucleic acid (PNA). In PNA compounds, thesugar backbone of an RNA is replaced with an amide containing backbone,in particular an aminoethylglycine backbone. The nucleobases areretained and are bound directly or indirectly to aza nitrogen atoms ofthe amide portion of the backbone. Representative U.S. patents thatteach the preparation of PNA compounds include, but are not limited to,U.S. Pat. Nos. 5,539,082; 5,714,331; and 5,719,262, the entire contentsof each of which are hereby incorporated herein by reference. AdditionalPNA compounds suitable for use in the iRNAs of the invention aredescribed in, for example, in Nielsen et al., Science, 1991, 254,1497-1500.

Some embodiments featured in the invention include RNAs withphosphorothioate backbones and oligonucleosides with heteroatombackbones, and in particular —CH₂—NH—CH₂—, —CH₂—N(CH₃)—O—CH₂—[known as amethylene (methylimino) or MMI backbone], —CH₂—O—N(CH₃)—CH₂—,—CH₂—N(CH₃)—N(CH₃)—CH₂— and —N(CH₃)—CH₂—CH₂—[wherein the nativephosphodiester backbone is represented as —O—P—O—CH₂—] of theabove-referenced U.S. Pat. No. 5,489,677, and the amide backbones of theabove-referenced U.S. Pat. No. 5,602,240. In some embodiments, the RNAsfeatured herein have morpholino backbone structures of theabove-referenced U.S. Pat. No. 5,034,506.

Modified RNAs can also contain one or more substituted sugar moieties.The iRNAs, e.g., dsRNAs, featured herein can include one of thefollowing at the 2′-position: OH; F; O-, S-, or N-alkyl; O-, S-, orN-alkenyl; O-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl,alkenyl and alkynyl can be substituted or unsubstituted C₁ to C₁₀ alkylor C₂ to C₁₀ alkenyl and alkynyl. Exemplary suitable modificationsinclude O[(CH₂)_(n)O]_(m)CH₃, O(CH₂)._(n)OCH₃, O(CH₂)_(n)NH₂,O(CH₂)_(n)CH₃, O(CH₂)_(n)ONH₂, and O(CH₂)_(n)ON[(CH₂)_(n)CH₃)]₂, where nand m are from 1 to about 10. In other embodiments, dsRNAs include oneof the following at the 2′ position: C₁ to C₁₀ lower alkyl, substitutedlower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH₃, OCN,Cl, Br, CN, CF₃, OCF₃, SOCH₃, SO₂CH₃, ONO₂, NO₂, N₃, NH₂,heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino,substituted silyl, an RNA cleaving group, a reporter group, anintercalator, a group for improving the pharmacokinetic properties of aniRNA, or a group for improving the pharmacodynamic properties of aniRNA, and other substituents having similar properties. In someembodiments, the modification includes a 2′-methoxyethoxy(2′-O—CH₂CH₂OCH₃, also known as 2′-O-(2-methoxyethyl) or 2′-MOE) (Martinet al., Helv. Chim. Acta, 1995, 78:486-504) i.e., an alkoxy-alkoxygroup. Another exemplary modification is 2′-dimethylaminooxyethoxy,i.e., a O(CH₂)₂ON(CH₃)₂ group, also known as 2′-DMAOE, as described inexamples herein below, and 2′-dimethylaminoethoxyethoxy (also known inthe art as 2′-O-dimethylaminoethoxyethyl or 2′-DMAEOE), i.e.,2′-O—CH₂—O—CH₂—N(CH₂)₂.

Other modifications include 2′-methoxy (2′-OCH₃), 2′-aminopropoxy(2′-OCH₂CH₂CH₂NH₂) and 2′-fluoro (2′-F). Similar modifications can alsobe made at other positions on the RNA of an iRNA, particularly the 3′position of the sugar on the 3′ terminal nucleotide or in 2′-5′ linkeddsRNAs and the 5′ position of 5′ terminal nucleotide. iRNAs can alsohave sugar mimetics such as cyclobutyl moieties in place of thepentofuranosyl sugar. Representative U.S. patents that teach thepreparation of such modified sugar structures include, but are notlimited to, U.S. Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044;5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811;5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873;5,646,265; 5,658,873; 5,670,633; and 5,700,920, certain of which arecommonly owned with the instant application. The entire contents of eachof the foregoing are hereby incorporated herein by reference.

An iRNA can also include nucleobase (often referred to in the art simplyas “base”) modifications or substitutions. As used herein, “unmodified”or “natural” nucleobases include the purine bases adenine (A) andguanine (G), and the pyrimidine bases thymine (T), cytosine (C) anduracil (U). Modified nucleobases include other synthetic and naturalnucleobases such as deoxy-thymine (dT), 5-methylcytosine (5-me-C),5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine,6-methyl and other alkyl derivatives of adenine and guanine, 2-propyland other alkyl derivatives of adenine and guanine, 2-thiouracil,2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyluracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil(pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl,8-hydroxyl anal other 8-substituted adenines and guanines, 5-halo,particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracilsand cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and8-azaadenine, 7-deazaguanine and 7-daazaadenine and 3-deazaguanine and3-deazaadenine. Further nucleobases include those disclosed in U.S. Pat.No. 3,687,808, those disclosed in Modified Nucleosides in Biochemistry,Biotechnology and Medicine, Herdewijn, P. ed. Wiley-VCH, 2008; thosedisclosed in The Concise Encyclopedia Of Polymer Science AndEngineering, pages 858-859, Kroschwitz, J. L, ed. John Wiley & Sons,1990, these disclosed by Englisch et al., Angewandte Chemie,International Edition, 1991, 30, 613, and those disclosed by Sanghvi, YS., Chapter 15, dsRNA Research and Applications, pages 289-302, Crooke,S. T. and Lebleu, B., Ed., CRC Press, 1993. Certain of these nucleobasesare particularly useful for increasing the binding affinity of theoligomeric compounds featured in the invention. These include5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6substituted purines, including 2-aminopropyladenine, 5-propynyluraciland 5-propynylcytosine. 5-methylcytosine substitutions have been shownto increase nucleic acid duplex stability by 0.6-1.2° C. (Sanghvi, Y.S., Crooke, S. T. and Lebleu, B., Eds., dsRNA Research and Applications,CRC Press, Boca Raton, 1993, pp. 276-278) and are exemplary basesubstitutions, even more particularly when combined with2′-O-methoxyethyl sugar modifications.

Representative U.S. patents that teach the preparation of certain of theabove noted modified nucleobases as well as other modified nucleobasesinclude, but are not limited to, the above noted U.S. Pat. Nos.3,687,808, 4,845,205; 5,130,30; 5,134,066; 5,175,273; 5,367,066;5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711;5,552,540; 5,587,469; 5,594,121, 5,596,091; 5,614,617; 5,681,941;5,750,692; 6,015,886; 6,147,200; 6,166,197; 6,222,025; 6,235,887;6,380,368; 6,528,640; 6,639,062; 6,617,438; 7,045,610; 7,427,672; and7,495,088, the entire contents of each of which are hereby incorporatedherein by reference.

The RNA of an iRNA can also be modified to include one or more bicyclicsugar moities. A “bicyclic sugar” is a furanosyl ring modified by thebridging of two atoms. A“bicyclic nucleoside” (“BNA”) is a nucleosidehaving a sugar moiety comprising a bridge connecting two carbon atoms ofthe sugar ring, thereby forming a bicyclic ring system. In certainembodiments, the bridge connects the 4′-carbon and the 2′-carbon of thesugar ring. Thus, in some embodiments an agent of the invention mayinclude one or more locked nucleic acids (LNA). A locked nucleic acid isa nucleotide having a modified ribose moiety in which the ribose moietycomprises an extra bridge connecting the 2′ and 4′ carbons. In otherwords, an LNA is a nucleotide comprising a bicyclic sugar moietycomprising a 4′-CH2-O-2′ bridge. This structure effectively “locks” theribose in the 3′-endo structural conformation. The addition of lockednucleic acids to siRNAs has been shown to increase siRNA stability inserum, and to reduce off-target effects (Elmen, J. et al., (2005)Nucleic Acids Research 33(1):439-447; Mook, O R. et al., (2007) Mol CancTher 6(3):833-843; Grunweller, A. et al., (2003) Nucleic Acids Research31(12):3185-3193). Examples of bicyclic nucleosides for use in thepolynucleotides of the invention include without limitation nucleosidescomprising a bridge between the 4′ and the 2′ ribosyl ring atoms. Incertain embodiments, the antisense polynucleotide agents of theinvention include one or more bicyclic nucleosides comprising a 4′ to 2′bridge. Examples of such 4′ to 2′ bridged bicyclic nucleosides, includebut are not limited to 4′-(CH2)-O-2′ (LNA); 4′-(CH2)-S-2′;4′-(CH2)2-O-2′ (ENA); 4′-CH(CH3)-O-2′ (also referred to as “constrainedethyl” or “cEt”) and 4′-CH(CH2OCH3)-O-2′ (and analogs thereof; see,e.g., U.S. Pat. No. 7,399,845); 4′-C(CH3)(CH3)-O-2′ (and analogsthereof; see e.g., U.S. Pat. No. 8,278,283); 4′-CH2-N(OCH3)-2′ (andanalogs thereof; see e.g., U.S. Pat. No. 8,278,425); 4′-CH2-O—N(CH3)-2′(see, e.g., U.S. Patent Publication No. 2004/0171570); 4′-CH2-N(R)—O-2′,wherein R is H, C1-C12 alkyl, or a protecting group (see, e.g., U.S.Pat. No. 7,427,672); 4′-CH2-C(H)(CH3)-2′ (see, e.g., Chattopadhyaya etal., J. Org. Chem., 2009, 74, 118-134); and 4′-CH2-C(═CH2)-2′ (andanalogs thereof; see, e.g., U.S. Pat. No. 8,278,426). The entirecontents of each of the foregoing are hereby incorporated herein byreference.

Additional representative U.S. patents and US Patent Publications thatteach the preparation of locked nucleic acid nucleotides include, butare not limited to, the following: U.S. Pat. Nos. 6,268,490; 6,525,191;6,670,461; 6,770,748; 6,794,499; 6,998,484; 7,053,207; 7,034,133;7,084,125; 7,399,845; 7,427,672; 7,569,686; 7,741,457; 8,022,193;8,030,467; 8,278,425; 8,278,426; 8,278,283; US 2008/0039618; and US2009/0012281, the entire contents of each of which are herebyincorporated herein by reference.

Any of the foregoing bicyclic nucleosides can be prepared having one ormore stereochemical sugar configurations including for exampleα-L-ribofuranose and β-D-ribofuranose (see WO 99/14226).

The RNA of an iRNA can also be modified to include one or moreconstrained ethyl nucleotides. As used herein, a “constrained ethylnucleotide” or “cEt” is a locked nucleic acid comprising a bicyclicsugar moiety comprising a 4′-CH(CH3)-0-2′ bridge. In one embodiment, aconstrained ethyl nucleotide is in the S conformation referred to hereinas “S-cEt.”

An iRNA of the invention may also include one or more “conformationallyrestricted nucleotides” (“CRN”). CRN are nucleotide analogs with alinker connecting the C2′ and C4′ carbons of ribose or the C3 and —C5′carbons of ribose. CRN lock the ribose ring into a stable conformationand increase the hybridization affinity to mRNA. The linker is ofsufficient length to place the oxygen in an optimal position forstability and affinity resulting in less ribose ring puckering.

Representative publications that teach the preparation of certain of theabove noted CRN include, but are not limited to, US Patent PublicationNo. 2013/0190383; and PCT publication WO 2013/036868, the entirecontents of each of which are hereby incorporated herein by reference.

One or more of the nucleotides of an iRNA of the invention may alsoinclude a hydroxymethyl substituted nucleotide. A “hydroxymethylsubstituted nucleotide” is an acyclic 2′-3′-seco-nucleotide, alsoreferred to as an “unlocked nucleic acid” (“UNA”) modification

Representative U.S. publications that teach the preparation of UNAinclude, but are not limited to, U.S. Pat. No. 8,314,227; and US PatentPublication Nos. 2013/0096289; 2013/0011922; and 2011/0313020, theentire contents of each of which are hereby incorporated herein byreference.

Potentially stabilizing modifications to the ends of RNA molecules caninclude N-(acetylaminocaproyl)-4-hydroxyprolinol (Hyp-C6-NHAc),N-(caproyl-4-hydroxyprolinol (Hyp-C6), N-(acetyl-4-hydroxyprolinol(Hyp-NHAc), thymidine-2′-0-deoxythymidine (ether),N-(aminocaproyl)-4-hydroxyprolinol (Hyp-C6-amino),2-docosanoyl-uridine-3″-phosphate, inverted base dT(idT) and others.Disclosure of this modification can be found in PCT Publication No. WO2011/005861.

Other modifications of the nucleotides of an iRNA of the inventioninclude a 5′ phosphate or 5′ phosphate mimic, e.g., a 5′-terminalphosphate or phosphate mimic on the antisense strand of an RNAi agent.Suitable phosphate mimics are disclosed in, for example US PatentPublication No. 2012/0157511, the entire contents of which areincorporated herein by reference.

A. Modified iRNAs Comprising Motifs of the Invention

In certain aspects of the invention, the double-stranded RNAi agents ofthe invention include agents with chemical modifications as disclosed,for example, in WO 2013/075035, filed on Nov. 16, 2012, the entirecontents of which are incorporated herein by reference. As shown hereinand in PCT Publication No. WO 2013/075035, a superior result may beobtained by introducing one or more motifs of three identicalmodifications on three consecutive nucleotides into a sense strandand/or antisense strand of an RNAi agent, particularly at or near thecleavage site. In some embodiments, the sense strand and antisensestrand of the RNAi agent may otherwise be completely modified. Theintroduction of these motifs interrupts the modification pattern, ifpresent, of the sense and/or antisense strand. The RNAi agent may beoptionally conjugated with a GalNAc derivative ligand, for instance onthe sense strand. The resulting RNAi agents present superior genesilencing activity.

More specifically, it has been surprisingly discovered that when thesense strand and antisense strand of the double-stranded RNAi agent arecompletely modified to have one or more motifs of three identicalmodifications on three consecutive nucleotides at or near the cleavagesite of at least one strand of an RNAi agent, the gene silencingactivity of the RNAi agent was superiorly enhanced.

Accordingly, the invention provides double-stranded RNAi agents capableof inhibiting the expression of a target gene (i.e., HDV gene) in vivo.The RNAi agent comprises a sense strand and an antisense strand. Eachstrand of the RNAi agent may range from 12-30 nucleotides in length. Forexample, each strand may be between 14-30 nucleotides in length, 17-30nucleotides in length, 25-30 nucleotides in length, 27-30 nucleotides inlength, 17-23 nucleotides in length, 17-21 nucleotides in length, 17-19nucleotides in length, 19-25 nucleotides in length, 19-23 nucleotides inlength, 19-21 nucleotides in length, 21-25 nucleotides in length, or21-23 nucleotides in length.

The sense strand and antisense strand typically form a duplex doublestranded RNA (“dsRNA”), also referred to herein as an “RNAi agent.” Theduplex region of an RNAi agent may be 12-30 nucleotide pairs in length.For example, the duplex region can be between 14-30 nucleotide pairs inlength, 17-30 nucleotide pairs in length, 27-30 nucleotide pairs inlength, 17-23 nucleotide pairs in length, 17-21 nucleotide pairs inlength, 17-19 nucleotide pairs in length, 19-25 nucleotide pairs inlength, 19-23 nucleotide pairs in length, 19-21 nucleotide pairs inlength, 21-25 nucleotide pairs in length, or 21-23 nucleotide pairs inlength. In another example, the duplex region is selected from 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, and 27 nucleotides in length.

In one embodiment, the RNAi agent may contain one or more overhangregions and/or capping groups at the 3′-end, 5′-end, or both ends of oneor both strands. The overhang can be 1-6 nucleotides in length, forinstance 2-6 nucleotides in length, 1-5 nucleotides in length, 2-5nucleotides in length, 1-4 nucleotides in length, 2-4 nucleotides inlength, 1-3 nucleotides in length, 2-3 nucleotides in length, or 1-2nucleotides in length. The overhangs can be the result of one strandbeing longer than the other, or the result of two strands of the samelength being staggered. The overhang can form a mismatch with the targetmRNA or it can be complementary to the gene sequences being targeted orcan be another sequence. The first and second strands can also bejoined, e.g., by additional bases to form a hairpin, or by othernon-base linkers.

In one embodiment, the nucleotides in the overhang region of the RNAiagent can each independently be a modified or unmodified nucleotideincluding, but no limited to 2′-sugar modified, such as, 2-F,2′-Omethyl, thymidine (T), 2′-O-methoxyethyl-5-methyluridine (Teo),2′-O-methoxyethyladenosine (Aeo), 2′-O-methoxyethyl-5-methylcytidine(m5Ceo), and any combinations thereof. For example, TT can be anoverhang sequence for either end on either strand. The overhang can forma mismatch with the target mRNA or it can be complementary to the genesequences being targeted or can be another sequence.

The 5′- or 3′-overhangs at the sense strand, antisense strand or bothstrands of the RNAi agent may be phosphorylated. In some embodiments,the overhang region(s) contains two nucleotides having aphosphorothioate between the two nucleotides, where the two nucleotidescan be the same or different. In one embodiment, the overhang is presentat the 3′-end of the sense strand, antisense strand, or both strands. Inone embodiment, this 3′-overhang is present in the antisense strand. Inone embodiment, this 3′-overhang is present in the sense strand.

The RNAi agent may contain only a single overhang, which can strengthenthe interference activity of the RNAi, without affecting its overallstability. For example, the single-stranded overhang may be located atthe 3′-terminal end of the sense strand or, alternatively, at the3′-terminal end of the antisense strand. The RNAi may also have a bluntend, located at the 5′-end of the antisense strand (or the 3′-end of thesense strand) or vice versa. Generally, the antisense strand of the RNAihas a nucleotide overhang at the 3′-end, and the 5′-end is blunt. Whilenot wishing to be bound by theory, the asymmetric blunt end at the5′-end of the antisense strand and 3′-end overhang of the antisensestrand favor the guide strand loading into RISC process.

In one embodiment, the RNAi agent is a double ended bluntmer of 19nucleotides in length, wherein the sense strand contains at least onemotif of three 2′-F modifications on three consecutive nucleotides atpositions 7, 8, 9 from the 5′end. The antisense strand contains at leastone motif of three 2′-O-methyl modifications on three consecutivenucleotides at positions 11, 12, 13 from the 5′end.

In another embodiment, the RNAi agent is a double ended bluntmer of 20nucleotides in length, wherein the sense strand contains at least onemotif of three 2′-F modifications on three consecutive nucleotides atpositions 8, 9, 10 from the 5′end. The antisense strand contains atleast one motif of three 2′-O-methyl modifications on three consecutivenucleotides at positions 11, 12, 13 from the 5′end.

In yet another embodiment, the RNAi agent is a double ended bluntmer of21 nucleotides in length, wherein the sense strand contains at least onemotif of three 2′-F modifications on three consecutive nucleotides atpositions 9, 10, 11 from the 5′end. The antisense strand contains atleast one motif of three 2′-O-methyl modifications on three consecutivenucleotides at positions 11, 12, 13 from the 5′end.

In one embodiment, the RNAi agent comprises a 21 nucleotide sense strandand a 23 nucleotide antisense strand, wherein the sense strand containsat least one motif of three 2′-F modifications on three consecutivenucleotides at positions 9, 10, 11 from the 5′end; the antisense strandcontains at least one motif of three 2′-O-methyl modifications on threeconsecutive nucleotides at positions 11, 12, 13 from the 5′end, whereinone end of the RNAi agent is blunt, while the other end comprises a 2nucleotide overhang. Preferably, the 2 nucleotide overhang is at the3′-end of the antisense strand.

When the 2 nucleotide overhang is at the 3′-end of the antisense strand,there may be two phosphorothioate internucleotide linkages between theterminal three nucleotides, wherein two of the three nucleotides are theoverhang nucleotides, and the third nucleotide is a paired nucleotidenext to the overhang nucleotide. In one embodiment, the RNAi agentadditionally has two phosphorothioate internucleotide linkages betweenthe terminal three nucleotides at both the 5′-end of the sense strandand at the 5′-end of the antisense strand. In one embodiment, everynucleotide in the sense strand and the antisense strand of the RNAiagent, including the nucleotides that are part of the motifs aremodified nucleotides. In one embodiment each residue is independentlymodified with a 2′-O-methyl or 3′-fluoro, e.g., in an alternating motif.Optionally, the RNAi agent further comprises a ligand (preferablyGalNAc₃).

In one embodiment, the RNAi agent comprises a sense and an antisensestrand, wherein the sense strand is 25-30 nucleotide residues in length,wherein starting from the 5′ terminal nucleotide (position 1) positions1 to 23 of the first strand comprise at least 8 ribonucleotides; theantisense strand is 36-66 nucleotide residues in length and, startingfrom the 3′ terminal nucleotide, comprises at least 8 ribonucleotides inthe positions paired with positions 1-23 of sense strand to form aduplex; wherein at least the 3′ terminal nucleotide of antisense strandis unpaired with sense strand, and up to 6 consecutive 3′ terminalnucleotides are unpaired with sense strand, thereby forming a 3′ singlestranded overhang of 1-6 nucleotides; wherein the 5′ terminus ofantisense strand comprises from 10-30 consecutive nucleotides which areunpaired with sense strand, thereby forming a 10-30 nucleotide singlestranded 5′ overhang; wherein at least the sense strand 5′ terminal and3′ terminal nucleotides are base paired with nucleotides of antisensestrand when sense and antisense strands are aligned for maximumcomplementarity, thereby forming a substantially duplexed region betweensense and antisense strands; and antisense strand is sufficientlycomplementary to a target RNA along at least 19 ribonucleotides ofantisense strand length to reduce target gene expression when the doublestranded nucleic acid is introduced into a mammalian cell; and whereinthe sense strand contains at least one motif of three 2′-F modificationson three consecutive nucleotides, where at least one of the motifsoccurs at or near the cleavage site. The antisense strand contains atleast one motif of three 2′-O-methyl modifications on three consecutivenucleotides at or near the cleavage site.

In one embodiment, the RNAi agent comprises sense and antisense strands,wherein the RNAi agent comprises a first strand having a length which isat least 25 and at most 29 nucleotides and a second strand having alength which is at most 30 nucleotides with at least one motif of three2′-O-methyl modifications on three consecutive nucleotides at position11, 12, 13 from the 5′ end; wherein the 3′ end of the first strand andthe 5′ end of the second strand form a blunt end and the second strandis 1-4 nucleotides longer at its 3′ end than the first strand, whereinthe duplex region region which is at least 25 nucleotides in length, andthe second strand is sufficiently complementary to a target mRNA alongat least 19 nucleotide of the second strand length to reduce target geneexpression when the RNAi agent is introduced into a mammalian cell, andwherein dicer cleavage of the RNAi agent preferentially results in ansiRNA comprising the 3′ end of the second strand, thereby reducingexpression of the target gene in the mammal. Optionally, the RNAi agentfurther comprises a ligand.

In one embodiment, the sense strand of the RNAi agent contains at leastone motif of three identical modifications on three consecutivenucleotides, where one of the motifs occurs at the cleavage site in thesense strand.

In one embodiment, the antisense strand of the RNAi agent can alsocontain at least one motif of three identical modifications on threeconsecutive nucleotides, where one of the motifs occurs at or near thecleavage site in the antisense strand

For an RNAi agent having a duplex region of 17-23 nucleotide in length,the cleavage site of the antisense strand is typically around the 10, 11and 12 positions from the 5′-end. Thus the motifs of three identicalmodifications may occur at the 9, 10, 11 positions; 10, 11, 12positions; 11, 12, 13 positions; 12, 13, 14 positions; or 13, 14, 15positions of the antisense strand, the count starting from the 1^(st)nucleotide from the 5′-end of the antisense strand, or, the countstarting from the 1^(st) paired nucleotide within the duplex region fromthe 5′-end of the antisense strand. The cleavage site in the antisensestrand may also change according to the length of the duplex region ofthe RNAi from the 5′-end.

The sense strand of the RNAi agent may contain at least one motif ofthree identical modifications on three consecutive nucleotides at thecleavage site of the strand; and the antisense strand may have at leastone motif of three identical modifications on three consecutivenucleotides at or near the cleavage site of the strand. When the sensestrand and the antisense strand form a dsRNA duplex, the sense strandand the antisense strand can be so aligned that one motif of the threenucleotides on the sense strand and one motif of the three nucleotideson the antisense strand have at least one nucleotide overlap, i.e., atleast one of the three nucleotides of the motif in the sense strandforms a base pair with at least one of the three nucleotides of themotif in the antisense strand. Alternatively, at least two nucleotidesmay overlap, or all three nucleotides may overlap.

In one embodiment, the sense strand of the RNAi agent may contain morethan one motif of three identical modifications on three consecutivenucleotides. The first motif may occur at or near the cleavage site ofthe strand and the other motifs may be a wing modification. The term“wing modification” herein refers to a motif occurring at anotherportion of the strand that is separated from the motif at or near thecleavage site of the same strand. The wing modification is eitheradjacent to the first motif or is separated by at least one or morenucleotides. When the motifs are immediately adjacent to each other thenthe chemistry of the motifs are distinct from each other and when themotifs are separated by one or more nucleotide than the chemistries canbe the same or different. Two or more wing modifications may be present.For instance, when two wing modifications are present, each wingmodification may occur at one end relative to the first motif which isat or near cleavage site or on either side of the lead motif.

Like the sense strand, the antisense strand of the RNAi agent maycontain more than one motifs of three identical modifications on threeconsecutive nucleotides, with at least one of the motifs occurring at ornear the cleavage site of the strand. This antisense strand may alsocontain one or more wing modifications in an alignment similar to thewing modifications that may be present on the sense strand.

In one embodiment, the wing modification on the sense strand orantisense strand of the RNAi agent typically does not include the firstone or two terminal nucleotides at the 3′-end, 5′-end or both ends ofthe strand.

In another embodiment, the wing modification on the sense strand orantisense strand of the RNAi agent typically does not include the firstone or two paired nucleotides within the duplex region at the 3′-end,5′-end or both ends of the strand.

When the sense strand and the antisense strand of the RNAi agent eachcontain at least one wing modification, the wing modifications may fallon the same end of the duplex region, and have an overlap of one, two orthree nucleotides.

When the sense strand and the antisense strand of the RNAi agent eachcontain at least two wing modifications, the sense strand and theantisense strand can be so aligned that two modifications each from onestrand fall on one end of the duplex region, having an overlap of one,two or three nucleotides; two modifications each from one strand fall onthe other end of the duplex region, having an overlap of one, two orthree nucleotides; two modifications one strand fall on each side of thelead motif, having an overlap of one, two or three nucleotides in theduplex region.

In one embodiment, every nucleotide in the sense strand and antisensestrand of the RNAi agent, including the nucleotides that are part of themotifs, may be modified. Each nucleotide may be modified with the sameor different modification which can include one or more alteration ofone or both of the non-linking phosphate oxygens and/or of one or moreof the linking phosphate oxygens; alteration of a constituent of theribose sugar, e.g., of the 2′ hydroxyl on the ribose sugar; wholesalereplacement of the phosphate moiety with “dephospho” linkers;modification or replacement of a naturally occurring base; andreplacement or modification of the ribose-phosphate backbone.

As nucleic acids are polymers of subunits, many of the modificationsoccur at a position which is repeated within a nucleic acid, e.g., amodification of a base, or a phosphate moiety, or a non-linking O of aphosphate moiety. In some cases the modification will occur at all ofthe subject positions in the nucleic acid but in many cases it will not.By way of example, a modification may only occur at a 3′ or 5′ terminalposition, may only occur in a terminal region, e.g., at a position on aterminal nucleotide or in the last 2, 3, 4, 5, or 10 nucleotides of astrand. A modification may occur in a double strand region, a singlestrand region, or in both. A modification may occur only in the doublestrand region of a RNA or may only occur in a single strand region of aRNA. For example, a phosphorothioate modification at a non-linking Oposition may only occur at one or both termini, may only occur in aterminal region, e.g., at a position on a terminal nucleotide or in thelast 2, 3, 4, 5, or 10 nucleotides of a strand, or may occur in doublestrand and single strand regions, particularly at termini. The 5′ end orends can be phosphorylated.

It may be possible, e.g., to enhance stability, to include particularbases in overhangs, or to include modified nucleotides or nucleotidesurrogates, in single strand overhangs, e.g., in a 5′ or 3′ overhang, orin both. For example, it can be desirable to include purine nucleotidesin overhangs. In some embodiments all or some of the bases in a 3′ or 5′overhang may be modified, e.g., with a modification described herein.Modifications can include, e.g., the use of modifications at the 2′position of the ribose sugar with modifications that are known in theart, e.g., the use of deoxyribonucleotides, 2′-deoxy-2′-fluoro (2′-F) or2′-O-methyl modified instead of the ribosugar of the nucleobase, andmodifications in the phosphate group, e.g., phosphorothioatemodifications. Overhangs need not be homologous with the targetsequence.

In one embodiment, each residue of the sense strand and antisense strandis independently modified with LNA, CRN, cET, UNA, HNA, CeNA,2′-methoxyethyl, 2′-O-methyl, 2′-O-allyl, 2′-C-allyl, 2′-deoxy,2′-hydroxyl, or 2′-fluoro. The strands can contain more than onemodification. In one embodiment, each residue of the sense strand andantisense strand is independently modified with 2′-O-methyl or2′-fluoro.

At least two different modifications are typically present on the sensestrand and antisense strand. Those two modifications may be the2′-O-methyl or 2′-fluoro modifications, or others.

In one embodiment, the N_(a) and/or N_(b) comprise modifications of analternating pattern. The term “alternating motif” as used herein refersto a motif having one or more modifications, each modification occurringon alternating nucleotides of one strand. The alternating nucleotide mayrefer to one per every other nucleotide or one per every threenucleotides, or a similar pattern. For example, if A, B and C eachrepresent one type of modification to the nucleotide, the alternatingmotif can be “ABABABABABAB . . . ,” “AABBAABBAABB . . . ,” “AABAABAABAAB. . . ,” “AAABAAABAAAB . . . ,” “AAABBBAAABBB . . . ,” or “ABCABCABCABC. . . ,” etc.

The type of modifications contained in the alternating motif may be thesame or different. For example, if A, B, C, D each represent one type ofmodification on the nucleotide, the alternating pattern, i.e.,modifications on every other nucleotide, may be the same, but each ofthe sense strand or antisense strand can be selected from severalpossibilities of modifications within the alternating motif such as“ABABAB . . . ”, “ACACAC . . . ” “BDBDBD . . . ” or “CDCDCD . . . ,”etc.

In one embodiment, the RNAi agent of the invention comprises themodification pattern for the alternating motif on the sense strandrelative to the modification pattern for the alternating motif on theantisense strand is shifted. The shift may be such that the modifiedgroup of nucleotides of the sense strand corresponds to a differentlymodified group of nucleotides of the antisense strand and vice versa.For example, the sense strand when paired with the antisense strand inthe dsRNA duplex, the alternating motif in the sense strand may startwith “ABABAB” from 5′-3′ of the strand and the alternating motif in theantisense strand may start with “BABABA” from 5′-3′ of the strand withinthe duplex region. As another example, the alternating motif in thesense strand may start with “AABBAABB” from 5′-3′ of the strand and thealternating motif in the antisense strand may start with “BBAABBAA” from5′-3′ of the strand within the duplex region, so that there is acomplete or partial shift of the modification patterns between the sensestrand and the antisense strand.

In one embodiment, the RNAi agent comprises the pattern of thealternating motif of 2′-O-methyl modification and 2′-F modification onthe sense strand initially has a shift relative to the pattern of thealternating motif of 2′-O-methyl modification and 2′-F modification onthe antisense strand initially, i.e., the 2′-O-methyl modifiednucleotide on the sense strand base pairs with a 2′-F modifiednucleotide on the antisense strand and vice versa. The 1 position of thesense strand may start with the 2′-F modification, and the 1 position ofthe antisense strand may start with the 2′-O-methyl modification.

The introduction of one or more motifs of three identical modificationson three consecutive nucleotides to the sense strand and/or antisensestrand interrupts the initial modification pattern present in the sensestrand and/or antisense strand. This interruption of the modificationpattern of the sense and/or antisense strand by introducing one or moremotifs of three identical modifications on three consecutive nucleotidesto the sense and/or antisense strand surprisingly enhances the genesilencing activity to the target gene.

In one embodiment, when the motif of three identical modifications onthree consecutive nucleotides is introduced to any of the strands, themodification of the nucleotide next to the motif is a differentmodification than the modification of the motif. For example, theportion of the sequence containing the motif is “ . . . N_(a)YYYN_(b) .. . ,” where “Y” represents the modification of the motif of threeidentical modifications on three consecutive nucleotide, and “N_(a)” and“N_(b)” represent a modification to the nucleotide next to the motif“YYY” that is different than the modification of Y, and where N_(a) andN_(b) can be the same or different modifications. Alternatively, N_(a)and/or N_(b) may be present or absent when there is a wing modificationpresent.

The RNAi agent may further comprise at least one phosphorothioate ormethylphosphonate internucleotide linkage. The phosphorothioate ormethylphosphonate internucleotide linkage modification may occur on anynucleotide of the sense strand or antisense strand or both strands inany position of the strand. For instance, the internucleotide linkagemodification may occur on every nucleotide on the sense strand and/orantisense strand; each internucleotide linkage modification may occur inan alternating pattern on the sense strand and/or antisense strand; orthe sense strand or antisense strand may contain both internucleotidelinkage modifications in an alternating pattern. The alternating patternof the internucleotide linkage modification on the sense strand may bethe same or different from the antisense strand, and the alternatingpattern of the internucleotide linkage modification on the sense strandmay have a shift relative to the alternating pattern of theinternucleotide linkage modification on the antisense strand. In oneembodiment, a double-stranded RNAi agent comprises 6-8phosphorothioateinternucleotide linkages. In one embodiment, the antisense strandcomprises two phosphorothioate internucleotide linkages at the5′-terminus and two phosphorothioate internucleotide linkages at the3′-terminus, and the sense strand comprises at least twophosphorothioate internucleotide linkages at either the 5′-terminus orthe 3′-terminus.

In one embodiment, the RNAi comprises a phosphorothioate ormethylphosphonate internucleotide linkage modification in the overhangregion. For example, the overhang region may contain two nucleotideshaving a phosphorothioate or methylphosphonate internucleotide linkagebetween the two nucleotides. Internucleotide linkage modifications alsomay be made to link the overhang nucleotides with the terminal pairednucleotides within the duplex region. For example, at least 2, 3, 4, orall the overhang nucleotides may be linked through phosphorothioate ormethylphosphonate internucleotide linkage, and optionally, there may beadditional phosphorothioate or methylphosphonate internucleotidelinkages linking the overhang nucleotide with a paired nucleotide thatis next to the overhang nucleotide. For instance, there may be at leasttwo phosphorothioate internucleotide linkages between the terminal threenucleotides, in which two of the three nucleotides are overhangnucleotides, and the third is a paired nucleotide next to the overhangnucleotide. These terminal three nucleotides may be at the 3′-end of theantisense strand, the 3′-end of the sense strand, the 5′-end of theantisense strand, and/or the 5′end of the antisense strand.

In one embodiment, the 2 nucleotide overhang is at the 3′-end of theantisense strand, and there are two phosphorothioate internucleotidelinkages between the terminal three nucleotides, wherein two of thethree nucleotides are the overhang nucleotides, and the third nucleotideis a paired nucleotide next to the overhang nucleotide. Optionally, theRNAi agent may additionally have two phosphorothioate internucleotidelinkages between the terminal three nucleotides at both the 5′-end ofthe sense strand and at the 5′-end of the antisense strand.

In one embodiment, the RNAi agent comprises mismatch(es) with thetarget, within the duplex, or combinations thereof. The mismatch mayoccur in the overhang region or the duplex region. The base pair may beranked on the basis of their propensity to promote dissociation ormelting (e.g., on the free energy of association or dissociation of aparticular pairing, the simplest approach is to examine the pairs on anindividual pair basis, though next neighbor or similar analysis can alsobe used). In terms of promoting dissociation: A:U is preferred over G:C;G:U is preferred over G:C; and I:C is preferred over G:C (I=inosine).Mismatches, e.g., non-canonical or other than canonical pairings (asdescribed elsewhere herein) are preferred over canonical (A:T, A:U, G:C)pairings; and pairings which include a universal base are preferred overcanonical pairings.

In one embodiment, the RNAi agent comprises at least one of the first 1,2, 3, 4, or 5 base pairs within the duplex regions from the 5′-end ofthe antisense strand independently selected from the group of: A:U, G:U,I:C, and mismatched pairs, e.g., non-canonical or other than canonicalpairings or pairings which include a universal base, to promote thedissociation of the antisense strand at the 5′-end of the duplex.

In one embodiment, the nucleotide at the 1 position within the duplexregion from the 5′-end in the antisense strand is selected from thegroup consisting of A, dA, dU, U, and dT. Alternatively, at least one ofthe first 1, 2 or 3 base pair within the duplex region from the 5′-endof the antisense strand is an AU base pair. For example, the first basepair within the duplex region from the 5′-end of the antisense strand isan AU base pair.

In another embodiment, the nucleotide at the 3′-end of the sense strandis deoxy-thymine (dT). In another embodiment, the nucleotide at the3′-end of the antisense strand is deoxy-thymine (dT). In one embodiment,there is a short sequence of deoxy-thymine nucleotides, for example, twodT nucleotides on the 3′-end of the sense and/or antisense strand.

In one embodiment, the sense strand sequence may be represented byformula (I):

(I) 5′ n_(p)-N_(a)-(XXX)_(i)-N_(b)-YYY-N_(b)-(ZZZ)_(j)-N_(a)-n_(q) 3′

wherein:

i and j are each independently 0 or 1;

p and q are each independently 0-6;

each N_(a) independently represents an oligonucleotide sequencecomprising 0-25 modified nucleotides, each sequence comprising at leasttwo differently modified nucleotides;

each N_(b) independently represents an oligonucleotide sequencecomprising 0-10 modified nucleotides;

each n_(p) and n_(q) independently represent an overhang nucleotide;

wherein Nb and Y do not have the same modification; and

XXX, YYY and ZZZ each independently represent one motif of threeidentical modifications on three consecutive nucleotides. Preferably YYYis all 2′-F modified nucleotides.

In one embodiment, the N_(a) and/or N_(b) comprise modifications ofalternating pattern.

In one embodiment, the YYY motif occurs at or near the cleavage site ofthe sense strand. For example, when the RNAi agent has a duplex regionof 17-23 nucleotides in length, the YYY motif can occur at or thevicinity of the cleavage site (e.g.: can occur at positions 6, 7, 8, 7,8, 9, 8, 9, 10, 9, 10, 11, 10, 11, 12 or 11, 12, 13) of—the sensestrand, the count starting from the 1^(st) nucleotide, from the 5′-end;or optionally, the count starting at the 1^(st) paired nucleotide withinthe duplex region, from the 5′-end.

In one embodiment, i is 1 and j is 0, or i is 0 and j is 1, or both iand j are 1. The sense strand can therefore be represented by thefollowing formulas:

(Ib) 5′ n_(p)-N_(a)-YYY-N_(b)-ZZZ-N_(a)-n_(q) 3′; (Ic)5′ n_(p)-N_(a)-XXX-N_(b)-YYY-N_(a)-n_(q) 3′; or (Id)5′ n_(p)-N_(a)-XXX-N_(b)-YYY-N_(b)-ZZZ-N_(a)-n_(q) 3′.

When the sense strand is represented by formula (Ib), N_(b) representsan oligonucleotide sequence comprising 0-10, 0-7, 0-5, 0-4, 0-2 or 0modified nucleotides. Each N_(a) independently can represent anoligonucleotide sequence comprising 2-20, 2-15, or 2-10 modifiednucleotides.

When the sense strand is represented as formula (Ic), N_(b) representsan oligonucleotide sequence comprising 0-10, 0-7, 0-10, 0-7, 0-5, 0-4,0-2 or 0 modified nucleotides. Each N_(a) can independently represent anoligonucleotide sequence comprising 2-20, 2-15, or 2-10 modifiednucleotides.

When the sense strand is represented as formula (Id), each N_(b)independently represents an oligonucleotide sequence comprising 0-10,0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. Preferably, N_(b) is 0, 1,2, 3, 4, 5 or 6 Each N_(a) can independently represent anoligonucleotide sequence comprising 2-20, 2-15, or 2-10 modifiednucleotides.

Each of X, Y and Z may be the same or different from each other.

In other embodiments, i is 0 and j is 0, and the sense strand may berepresented by the formula:

(Ia) 5′ n_(p)-N_(a)-YYY-N_(a)-n_(q) 3′.

When the sense strand is represented by formula (Ia), each N_(a)independently can represent an oligonucleotide sequence comprising 2-20,2-15, or 2-10 modified nucleotides.

In one embodiment, the antisense strand sequence of the RNAi may berepresented by formula (II):

(II)5′ n_(q′)-N_(a)′-(Z′Z′Z′)_(k)-N_(b)′-Y′Y′Y′-N_(b)′-(X′X′X′)₁-N′_(a)-n_(p)′ 3'

wherein:

k and l are each independently 0 or 1;

p′ and q′ are each independently 0-6;

each N_(a)′ independently represents an oligonucleotide sequencecomprising 0-25 modified nucleotides, each sequence comprising at leasttwo differently modified nucleotides;

each N_(b)′ independently represents an oligonucleotide sequencecomprising 0-10 modified nucleotides;

each n_(p)′ and n_(q)′ independently represent an overhang nucleotide;

wherein N_(b)′ and Y′ do not have the same modification; and

X′X′X′, Y′Y′Y′ and Z′Z′Z′ each independently represent one motif ofthree identical modifications on three consecutive nucleotides.

In one embodiment, the N_(a)′ and/or N_(b)′ comprise modifications ofalternating pattern.

The Y′Y′Y′ motif occurs at or near the cleavage site of the antisensestrand. For example, when the RNAi agent has a duplex region of 17-23nucleotide in length, the Y′Y′Y′ motif can occur at positions 9, 10, 11;10, 11, 12; 11, 12, 13; 12, 13, 14; or 13, 14, 15 of the antisensestrand, with the count starting from the 1^(st) nucleotide, from the5′-end; or optionally, the count starting at the 1^(st) pairednucleotide within the duplex region, from the 5′-end. Preferably, theY′Y′Y′ motif occurs at positions 11, 12, 13.

In one embodiment, Y′Y′Y′ motif is all 2′-OMe modified nucleotides.

In one embodiment, k is 1 and l is 0, or k is 0 and l is 1, or both kand l are 1.

The antisense strand can therefore be represented by the followingformulas:

(IIb) 5′ n_(q′)-N_(a)′-Z′Z′Z′-N_(b)′-Y′Y′Y′-N_(a)′-n_(p′) 3′; (IIc)5′ n_(q′)-N_(a)′-Y′Y′Y′-N_(b)′-X′X′X′-n_(p′)3′; or (IId)5′ n_(q′)-N_(a)′-Z′Z′Z′-N_(b)′-Y′Y′Y′-N_(b)′-X′X′X′-N_(a)′-n_(p′) 3′.

When the antisense strand is represented by formula (IIb), N_(b)′represents an oligonucleotide sequence comprising 0-10, 0-7, 0-10, 0-7,0-5, 0-4, 0-2 or 0 modified nucleotides. Each N_(a)′ independentlyrepresents an oligonucleotide sequence comprising 2-20, 2-15, or 2-10modified nucleotides.

When the antisense strand is represented as formula (IIc), N_(b)′represents an oligonucleotide sequence comprising 0-10, 0-7, 0-10, 0-7,0-5, 0-4, 0-2 or 0 modified nucleotides. Each N_(a)′ independentlyrepresents an oligonucleotide sequence comprising 2-20, 2-15, or 2-10modified nucleotides.

When the antisense strand is represented as formula (IId), each N_(b)′independently represents an oligonucleotide sequence comprising 0-10,0-7, 0-10, 0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. Each N_(a)′independently represents an oligonucleotide sequence comprising 2-20,2-15, or 2-10 modified nucleotides. Preferably, N_(b) is 0, 1, 2, 3, 4,5 or 6.

In other embodiments, k is 0 and l is 0 and the antisense strand may berepresented by the formula:

(Ia) 5′ n_(p′)-N_(a′)-Y′Y′Y′-N_(a′)-n_(q′) 3′.

When the antisense strand is represented as formula (IIa), each N_(a)′independently represents an oligonucleotide sequence comprising 2-20,2-15, or 2-10 modified nucleotides.

Each of X′, Y′ and Z′ may be the same or different from each other.Each nucleotide of the sense strand and antisense strand may beindependently modified with LNA, CRN, UNA, cEt, HNA, CeNA,2′-methoxyethyl, 2′-O-methyl, 2′-O-allyl, 2′-C-allyl, 2′-hydroxyl, or2′-fluoro. For example, each nucleotide of the sense strand andantisense strand is independently modified with 2′-O-methyl or2′-fluoro. Each X, Y, Z, X′, Y′ and Z′, in particular, may represent a2′-O-methyl modification or a 2′-fluoro modification.

In one embodiment, the sense strand of the RNAi agent may contain YYYmotif occurring at 9, 10 and 11 positions of the strand when the duplexregion is 21 nt, the count starting from the 1^(st) nucleotide from the5′-end, or optionally, the count starting at the 1^(st) pairednucleotide within the duplex region, from the 5′-end; and Y represents2′-F modification. The sense strand may additionally contain XXX motifor ZZZ motifs as wing modifications at the opposite end of the duplexregion; and XXX and ZZZ each independently represents a 2′-OMemodification or 2′-F modification.

In one embodiment the antisense strand may contain Y′Y′Y′ motifoccurring at positions 11, 12, 13 of the strand, the count starting fromthe 1^(st) nucleotide from the 5′-end, or optionally, the count startingat the 1^(st) paired nucleotide within the duplex region, from the5′-end; and Y′ represents 2′-O-methyl modification. The antisense strandmay additionally contain X′X′X′ motif or Z′Z′Z′ motifs as wingmodifications at the opposite end of the duplex region; and X′X′X′ andZ′Z′Z′ each independently represents a 2′-OMe modification or 2′-Fmodification.

The sense strand represented by any one of the above formulas (Ia),(Ib), (Ic), and (Id) forms a duplex with a antisense strand beingrepresented by any one of formulas (IIa), (IIb), (IIc), and (IId),respectively.

Accordingly, the RNAi agents for use in the methods of the invention maycomprise a sense strand and an antisense strand, each strand having 14to 30 nucleotides, the RNAi duplex represented by formula (III):

(III) sense:5′ n_(p)-N_(a)-(XXX)_(i)-N_(b)-YYY-N_(b)-(ZZZ)_(j)-N_(a)-n_(q) 3'antisense:3′ n_(p)′-N_(a)′-(X′X′X′)_(k)-N_(b)′-Y′Y′Y′-N_(b)′-(Z′Z′Z′)₁-N_(a)′-n_(q)′ 5′

wherein:

i, j, k, and l are each independently 0 or 1;

p, p′, q, and q′ are each independently 0-6;

each N_(a) and N_(a)′ independently represents an oligonucleotidesequence comprising 0-25 modified nucleotides, each sequence comprisingat least two differently modified nucleotides;

each N_(b) and N_(b)′ independently represents an oligonucleotidesequence comprising 0-10 modified nucleotides;

wherein each n_(p)′, n_(p), n_(q)′, and n_(q), each of which may or maynot be present, independently represents an overhang nucleotide; and

XXX, YYY, ZZZ, X′X′X′, Y′Y′Y′, and Z′Z′Z′ each independently representone motif of three identical modifications on three consecutivenucleotides.

In one embodiment, i is 0 and j is 0; or i is 1 and j is 0; or i is 0and j is 1; or both i and j are 0; or both i and j are 1. In anotherembodiment, k is 0 and l is 0; or k is 1 and l is 0; k is 0 and l is 1;or both k and l are 0; or both k and l are 1.

Exemplary combinations of the sense strand and antisense strand forminga RNAi duplex include the formulas below:

(IIIa) 5′ n_(p)-N_(a)-YYY-N_(a)-n_(q) 3′3′ n_(p)′-N_(a)′-Y′Y′Y′-N_(a)′n_(q)′ 5′ (IIIb)5′ n_(p)-N_(a)-YYY-N_(b)-ZZZ-N_(a)-n_(q) 3′3′ n_(p)′-N_(a)′-Y′Y′Y′-N_(b)'-Z′Z′Z′-N_(a)′n_(q)′ 5′ (IIIc)5′ n_(p)-N_(a)-XXX-N_(b)-YYY-N_(a)-n_(q) 3′3′ n_(p)′-N_(a)′-X′X′X′-N_(b)′-Y′Y′Y′-N_(a)′-n_(q)′ 5′ (IIId)5′ n_(p)-N_(a)-XXX-N_(b)-YYY-N_(b)-ZZZ-N_(a)-n_(q) 3'3′ n_(p)′-N_(a)′-X′X′X′-N_(b)′-Y′Y′Y′-N_(b)′-Z′Z′Z′-N_(a)-n_(q)′ 5′

When the RNAi agent is represented by formula (IIIa), each N_(a)independently represents an oligonucleotide sequence comprising 2-20,2-15, or 2-10 modified nucleotides.

When the RNAi agent is represented by formula (IIIb), each N_(b)independently represents an oligonucleotide sequence comprising 1-10,1-7, 1-5 or 1-4 modified nucleotides. Each N_(a) independentlyrepresents an oligonucleotide sequence comprising 2-20, 2-15, or 2-10modified nucleotides.

When the RNAi agent is represented as formula (IIIc), each N_(b), N_(b)′independently represents an oligonucleotide sequence comprising 0-10,0-7, 0-10, 0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. Each N_(a)independently represents an oligonucleotide sequence comprising 2-20,2-15, or 2-10 modified nucleotides.

When the RNAi agent is represented as formula (IIId), each N_(b), N_(b)′independently represents an oligonucleotide sequence comprising 0-10,0-7, 0-10, 0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. Each N_(a),N_(a)′ independently represents an oligonucleotide sequence comprising2-20, 2-15, or 2-10 modified nucleotides. Each of N_(a), N_(a)′, N_(b)and N_(b)′ independently comprises modifications of alternating pattern.

Each of X, Y and Z in formulas (III), (IIIa), (IIIb), (IIIc), and (IIId)may be the same or different from each other.

When the RNAi agent is represented by formula (III), (IIIa), (IIIb),(IIIc), and (IIId), at least one of the Y nucleotides may form a basepair with one of the Y′ nucleotides. Alternatively, at least two of theY nucleotides form base pairs with the corresponding Y′ nucleotides; orall three of the Y nucleotides all form base pairs with thecorresponding Y′ nucleotides.

When the RNAi agent is represented by formula (IIIb) or (IIId), at leastone of the Z nucleotides may form a base pair with one of the Z′nucleotides. Alternatively, at least two of the Z nucleotides form basepairs with the corresponding Z′ nucleotides; or all three of the Znucleotides all form base pairs with the corresponding Z′ nucleotides.

When the RNAi agent is represented as formula (IIIc) or (IIId), at leastone of the X nucleotides may form a base pair with one of the X′nucleotides. Alternatively, at least two of the X nucleotides form basepairs with the corresponding X′ nucleotides; or all three of the Xnucleotides all form base pairs with the corresponding X′ nucleotides.

In one embodiment, the modification on the Y nucleotide is differentthan the modification on the Y′ nucleotide, the modification on the Znucleotide is different than the modification on the Z′ nucleotide,and/or the modification on the X nucleotide is different than themodification on the X′ nucleotide.

In one embodiment, when the RNAi agent is represented by formula (IIId),the N_(a) modifications are 2′-O-methyl or 2′-fluoro modifications. Inanother embodiment, when the RNAi agent is represented by formula(IIId), the N_(a) modifications are 2′-O-methyl or 2′-fluoromodifications and n_(p)′>0 and at least one n_(p)′ is linked to aneighboring nucleotide a via phosphorothioate linkage. In yet anotherembodiment, when the RNAi agent is represented by formula (IIId), theN_(a) modifications are 2′-O-methyl or 2′-fluoro modifications, n_(p)′>0and at least one n_(p)′ is linked to a neighboring nucleotide viaphosphorothioate linkage, and the sense strand is conjugated to one ormore GalNAc derivatives attached through a bivalent or trivalentbranched linker (described below). In another embodiment, when the RNAiagent is represented by formula (IIId), the N_(a) modifications are2′-O-methyl or 2′-fluoro modifications, n_(p)′>0 and at least one n_(p)′is linked to a neighboring nucleotide via phosphorothioate linkage, thesense strand comprises at least one phosphorothioate linkage, and thesense strand is conjugated to one or more GalNAc derivatives attachedthrough a bivalent or trivalent branched linker.

In one embodiment, when the RNAi agent is represented by formula (IIIa),the N_(a) modifications are 2′-O-methyl or 2′-fluoro modifications,n_(p)′>0 and at least one n_(p)′ is linked to a neighboring nucleotidevia phosphorothioate linkage, the sense strand comprises at least onephosphorothioate linkage, and the sense strand is conjugated to one ormore GalNAc derivatives attached through a bivalent or trivalentbranched linker.

In one embodiment, the RNAi agent is a multimer containing at least twoduplexes represented by formula (III), (IIIa), (IIIb), (IIIc), and(IIId), wherein the duplexes are connected by a linker. The linker canbe cleavable or non-cleavable. Optionally, the multimer furthercomprises a ligand. Each of the duplexes can target the same gene or twodifferent genes; or each of the duplexes can target same gene at twodifferent target sites.

In one embodiment, the RNAi agent is a multimer containing three, four,five, six or more duplexes represented by formula (III), (IIIa), (IIIb),(IIIc), and (IIId), wherein the duplexes are connected by a linker. Thelinker can be cleavable or non-cleavable. Optionally, the multimerfurther comprises a ligand. Each of the duplexes can target the samegene or two different genes; or each of the duplexes can target samegene at two different target sites.

In one embodiment, two RNAi agents represented by formula (III), (IIIa),(IIIb), (IIIc), and (IIId) are linked to each other at the 5′ end, andone or both of the 3′ ends and are optionally conjugated to to a ligand.Each of the agents can target the same gene or two different genes; oreach of the agents can target same gene at two different target sites.

Various publications describe multimeric RNAi agents that can be used inthe methods of the invention. Such publications include WO2007/091269,U.S. Pat. No. 7,858,769, WO2010/141511, WO2007/117686, WO2009/014887 andWO2011/031520 the entire contents of each of which are herebyincorporated herein by reference.

As described in more detail below, the RNAi agent that containsconjugations of one or more carbohydrate moieties to a RNAi agent canoptimize one or more properties of the RNAi agent. In many cases, thecarbohydrate moiety will be attached to a modified subunit of the RNAiagent. For example, the ribose sugar of one or more ribonucleotidesubunits of a dsRNA agent can be replaced with another moiety, e.g., anon-carbohydrate (preferably cyclic) carrier to which is attached acarbohydrate ligand. A ribonucleotide subunit in which the ribose sugarof the subunit has been so replaced is referred to herein as a ribosereplacement modification subunit (RRMS). A cyclic carrier may be acarbocyclic ring system, i.e., all ring atoms are carbon atoms, or aheterocyclic ring system, i.e., one or more ring atoms may be aheteroatom, e.g., nitrogen, oxygen, sulfur. The cyclic carrier may be amonocyclic ring system, or may contain two or more rings, e.g. fusedrings. The cyclic carrier may be a fully saturated ring system, or itmay contain one or more double bonds.

The ligand may be attached to the polynucleotide via a carrier. Thecarriers include (i) at least one “backbone attachment point,”preferably two “backbone attachment points” and (ii) at least one“tethering attachment point.” A “backbone attachment point” as usedherein refers to a functional group, e.g. a hydroxyl group, orgenerally, a bond available for, and that is suitable for incorporationof the carrier into the backbone, e.g., the phosphate, or modifiedphosphate, e.g., sulfur containing, backbone, of a ribonucleic acid. A“tethering attachment point” (TAP) in some embodiments refers to aconstituent ring atom of the cyclic carrier, e.g., a carbon atom or aheteroatom (distinct from an atom which provides a backbone attachmentpoint), that connects a selected moiety. The moiety can be, e.g., acarbohydrate, e.g. monosaccharide, disaccharide, trisaccharide,tetrasaccharide, oligosaccharide and polysaccharide. Optionally, theselected moiety is connected by an intervening tether to the cycliccarrier. Thus, the cyclic carrier will often include a functional group,e.g., an amino group, or generally, provide a bond, that is suitable forincorporation or tethering of another chemical entity, e.g., a ligand tothe constituent ring.

The RNAi agents may be conjugated to a ligand via a carrier, wherein thecarrier can be cyclic group or acyclic group; preferably, the cyclicgroup is selected from pyrrolidinyl, pyrazolinyl, pyrazolidinyl,imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3]dioxolane,oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl,isothiazolidinyl, quinoxalinyl, pyridazinonyl, tetrahydrofuryl and anddecalin; preferably, the acyclic group is selected from serinol backboneor diethanolamine backbone.

In certain specific embodiments, the RNAi agent for use in the methodsof the invention is an agent selected from the group of agents listed inany one of Tables 11, 12, 31, and 32. These agents may further comprisea ligand. In one embodiment, the agent is selected from the groupconsisting of AD-70260.1, AD-70232.1, AD-70249.1, AD-70244.1,AD-70272.1, AD-70228.1, AD-70255.1, AD-70278.1, AD-70295.1, AD-67200.1,AD-67211.1, AD-67199.1, AD-67202.1, AD-67208.1, AD-67210.1, AD-70259.1,AD-70267.1, AD-70272.1, AD-70271.1, AD-70268.1, AD-70269.1, AD-70232.1,AD-70256.1, AD-70257.1, or AD-70275.1. In another embodiment, the agentis selected from the group consisting of AD-70260.1, AD-70232.1,AD-70249.1, AD-70244.1, AD-70272.1, AD-70228.1, AD-70255.1, AD-70278.1,or AD-70295.1.

IV. iRNAs Conjugated to Ligands

Another modification of the RNA of an iRNA of the invention involveschemically linking to the RNA one or more ligands, moieties orconjugates that enhance the activity, cellular distribution or cellularuptake of the iRNA. Such moieties include but are not limited to lipidmoieties such as a cholesterol moiety (Letsinger et al., Proc. Natl.Acid. Sci. USA, 1989, 86: 6553-6556), cholic acid (Manoharan et al.,Biorg. Med. Chem. Let., 1994, 4:1053-1060), a thioether, e.g.,beryl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992,660:306-309; Manoharan et al., Biorg. Med. Chem. Let., 1993,3:2765-2770), a thiocholesterol (Oberhauser et al., Nucl. Acids Res.,1992, 20:533-538), an aliphatic chain, e.g., dodecandiol or undecylresidues (Saison-Behmoaras et al., EMBO J, 1991, 10:1111-1118; Kabanovet al., FEBS Lett., 1990, 259:327-330; Svinarchuk et al., Biochimie,1993, 75:49-54), a phospholipid, e.g., di-hexadecyl-rac-glycerol ortriethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-phosphonate (Manoharanet al., Tetrahedron Lett., 1995, 36:3651-3654; Shea et al., Nucl. AcidsRes., 1990, 18:3777-3783), a polyamine or a polyethylene glycol chain(Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969-973), oradamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995,36:3651-3654), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta,1995, 1264:229-237), or an octadecylamine orhexylamino-carbonyloxycholesterol moiety (Crooke et al., J. Pharmacol.Exp. Ther., 1996, 277:923-937).

In one embodiment, a ligand alters the distribution, targeting orlifetime of an iRNA agent into which it is incorporated. In preferredembodiments a ligand provides an enhanced affinity for a selectedtarget, e.g., molecule, cell or cell type, compartment, e.g., a cellularor organ compartment, tissue, organ or region of the body, as, e.g.,compared to a species absent such a ligand. Preferred ligands will nottake part in duplex pairing in a duplexed nucleic acid.

Ligands can include a naturally occurring substance, such as a protein(e.g., human serum albumin (HSA), low-density lipoprotein (LDL), orglobulin); carbohydrate (e.g., a dextran, pullulan, chitin, chitosan,inulin, cyclodextrin, N-acetylgalactosamine, or hyaluronic acid); or alipid. The ligand can also be a recombinant or synthetic molecule, suchas a synthetic polymer, e.g., a synthetic polyamino acid. Examples ofpolyamino acids include polyamino acid is a polylysine (PLL), polyL-aspartic acid, poly L-glutamic acid, styrene-maleic acid anhydridecopolymer, poly(L-lactide-co-glycolide) copolymer, divinyl ether-maleicanhydride copolymer, N-(2-hydroxypropyl)methacrylamide copolymer (HMPA),polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane,poly(2-ethylacryllic acid), N-isopropylacrylamide polymers, orpolyphosphazine. Example of polyamines include: polyethylenimine,polylysine (PLL), spermine, spermidine, polyamine,pseudopeptide-polyamine, peptidomimetic polyamine, dendrimer polyamine,arginine, amidine, protamine, cationic lipid, cationic porphyrin,quaternary salt of a polyamine, or an alpha helical peptide.

Ligands can also include targeting groups, e.g., a cell or tissuetargeting agent, e.g., a lectin, glycoprotein, lipid or protein, e.g.,an antibody, that binds to a specified cell type such as a kidney cell.A targeting group can be a thyrotropin, melanotropin, lectin,glycoprotein, surfactant protein A, Mucin carbohydrate, multivalentlactose, multivalent galactose, N-acetyl-galactosamine,N-acetyl-gulucoseamine multivalent mannose, multivalent fucose,glycosylated polyaminoacids, multivalent galactose, transferrin,bisphosphonate, polyglutamate, polyaspartate, a lipid, cholesterol, asteroid, bile acid, folate, vitamin B12, vitamin A, biotin, or an RGDpeptide or RGD peptide mimetic.

Other examples of ligands include dyes, intercalating agents (e.g.acridines), cross-linkers (e.g. psoralene, mitomycin C), porphyrins(TPPC4, texaphyrin, Sapphyrin), polycyclic aromatic hydrocarbons (e.g.,phenazine, dihydrophenazine), artificial endonucleases (e.g. EDTA),lipophilic molecules, e.g., cholesterol, cholic acid, adamantane aceticacid, 1-pyrene butyric acid, dihydrotestosterone,1,3-Bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol,borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid,myristic acid,O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid,dimethoxytrityl, or phenoxazine) and peptide conjugates (e.g.,antennapedia peptide, Tat peptide), alkylating agents, phosphate, amino,mercapto, PEG (e.g., PEG-40K), MPEG, [MPEG]₂, polyamino, alkyl,substituted alkyl, radiolabeled markers, enzymes, haptens (e.g. biotin),transport/absorption facilitators (e.g., aspirin, vitamin E, folicacid), synthetic ribonucleases (e.g., imidazole, bisimidazole,histamine, imidazole clusters, acridine-imidazole conjugates, Eu3+complexes of tetraazamacrocycles), dinitrophenyl, HRP, or AP.

Ligands can be proteins, e.g., glycoproteins, or peptides, e.g.,molecules having a specific affinity for a co-ligand, or antibodiese.g., an antibody, that binds to a specified cell type such as a hepaticcell. Ligands can also include hormones and hormone receptors. They canalso include non-peptidic species, such as lipids, lectins,carbohydrates, vitamins, cofactors, multivalent lactose, multivalentgalactose, N-acetyl-galactosamine, N-acetyl-gulucosamine multivalentmannose, or multivalent fucose. The ligand can be, for example, alipopolysaccharide, an activator of p38 MAP kinase, or an activator ofNF-κB.

The ligand can be a substance, e.g., a drug, which can increase theuptake of the iRNA agent into the cell, for example, by disrupting thecell's cytoskeleton, e.g., by disrupting the cell's microtubules,microfilaments, and/or intermediate filaments. The drug can be, forexample, taxon, vincristine, vinblastine, cytochalasin, nocodazole,japlakinolide, latrunculin A, phalloidin, swinholide A, indanocine, ormyoservin.

In some embodiments, a ligand attached to an iRNA as described hereinacts as a pharmacokinetic modulator (PK modulator). PK modulatorsinclude lipophiles, bile acids, steroids, phospholipid analogues,peptides, protein binding agents, PEG, vitamins etc. Exemplary PKmodulators include, but are not limited to, cholesterol, fatty acids,cholic acid, lithocholic acid, dialkylglycerides, diacylglyceride,phospholipids, sphingolipids, naproxen, ibuprofen, vitamin E, biotinetc. Oligonucleotides that comprise a number of phosphorothioatelinkages are also known to bind to serum protein, thus shortoligonucleotides, e.g., oligonucleotides of about 5 bases, 10 bases, 15bases or 20 bases, comprising multiple of phosphorothioate linkages inthe backbone are also amenable to the present invention as ligands (e.g.as PK modulating ligands). In addition, aptamers that bind serumcomponents (e.g. serum proteins) are also suitable for use as PKmodulating ligands in the embodiments described herein.

Ligand-conjugated oligonucleotides of the invention may be synthesizedby the use of an oligonucleotide that bears a pendant reactivefunctionality, such as that derived from the attachment of a linkingmolecule onto the oligonucleotide (described below). This reactiveoligonucleotide may be reacted directly with commercially-availableligands, ligands that are synthesized bearing any of a variety ofprotecting groups, or ligands that have a linking moiety attachedthereto.

The oligonucleotides used in the conjugates of the present invention maybe conveniently and routinely made through the well-known technique ofsolid-phase synthesis. Equipment for such synthesis is sold by severalvendors including, for example, Applied Biosystems (Foster City,Calif.). Any other means for such synthesis known in the art mayadditionally or alternatively be employed. It is also known to usesimilar techniques to prepare other oligonucleotides, such as thephosphorothioates and alkylated derivatives.

In the ligand-conjugated oligonucleotides and ligand-molecule bearingsequence-specific linked nucleosides of the present invention, theoligonucleotides and oligonucleosides may be assembled on a suitable DNAsynthesizer utilizing standard nucleotide or nucleoside precursors, ornucleotide or nucleoside conjugate precursors that already bear thelinking moiety, ligand-nucleotide or nucleoside-conjugate precursorsthat already bear the ligand molecule, or non-nucleoside ligand-bearingbuilding blocks.

When using nucleotide-conjugate precursors that already bear a linkingmoiety, the synthesis of the sequence-specific linked nucleosides istypically completed, and the ligand molecule is then reacted with thelinking moiety to form the ligand-conjugated oligonucleotide. In someembodiments, the oligonucleotides or linked nucleosides of the presentinvention are synthesized by an automated synthesizer usingphosphoramidites derived from ligand-nucleoside conjugates in additionto the standard phosphoramidites and non-standard phosphoramidites thatare commercially available and routinely used in oligonucleotidesynthesis.

A. Lipid Conjugates

In one embodiment, the ligand or conjugate is a lipid or lipid-basedmolecule. Such a lipid or lipid-based molecule preferably binds a serumprotein, e.g., human serum albumin (HSA). An HSA binding ligand allowsfor distribution of the conjugate to a target tissue, e.g., a non-kidneytarget tissue of the body. For example, the target tissue can be theliver, including parenchymal cells of the liver. Other molecules thatcan bind HSA can also be used as ligands. For example, naproxen oraspirin can be used. A lipid or lipid-based ligand can (a) increaseresistance to degradation of the conjugate, (b) increase targeting ortransport into a target cell or cell membrane, and/or (c) can be used toadjust binding to a serum protein, e.g., HSA.

A lipid based ligand can be used to inhibit, e.g., control the bindingof the conjugate to a target tissue. For example, a lipid or lipid-basedligand that binds to HSA more strongly will be less likely to betargeted to the kidney and therefore less likely to be cleared from thebody. A lipid or lipid-based ligand that binds to HSA less strongly canbe used to target the conjugate to the kidney.

In a preferred embodiment, the lipid based ligand binds HSA. Preferably,it binds HSA with a sufficient affinity such that the conjugate will bepreferably distributed to a non-kidney tissue. However, it is preferredthat the affinity not be so strong that the HSA-ligand binding cannot bereversed.

In another preferred embodiment, the lipid based ligand binds HSA weaklyor not at all, such that the conjugate will be preferably distributed tothe kidney. Other moieties that target to kidney cells can also be usedin place of or in addition to the lipid based ligand.

In another aspect, the ligand is a moiety, e.g., a vitamin, which istaken up by a target cell, e.g., a proliferating cell. These areparticularly useful for treating disorders characterized by unwantedcell proliferation, e.g., of the malignant or non-malignant type, e.g.,cancer cells. Exemplary vitamins include vitamin A, E, and K. Otherexemplary vitamins include are B vitamin, e.g., folic acid, B12,riboflavin, biotin, pyridoxal or other vitamins or nutrients taken up bytarget cells such as liver cells. Also included are HSA and low densitylipoprotein (LDL).

B. Cell Permeation Agents

In another aspect, the ligand is a cell-permeation agent, preferably ahelical cell-permeation agent. Preferably, the agent is amphipathic. Anexemplary agent is a peptide such as tat or antennopedia. If the agentis a peptide, it can be modified, including a peptidylmimetic,invertomers, non-peptide or pseudo-peptide linkages, and use of D-aminoacids. The helical agent is preferably an alpha-helical agent, whichpreferably has a lipophilic and a lipophobic phase.

The ligand can be a peptide or peptidomimetic. A peptidomimetic (alsoreferred to herein as an oligopeptidomimetic) is a molecule capable offolding into a defined three-dimensional structure similar to a naturalpeptide. The attachment of peptide and peptidomimetics to iRNA agentscan affect pharmacokinetic distribution of the iRNA, such as byenhancing cellular recognition and absorption. The peptide orpeptidomimetic moiety can be about 5-50 amino acids long, e.g., about 5,10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids long.

A peptide or peptidomimetic can be, for example, a cell permeationpeptide, cationic peptide, amphipathic peptide, or hydrophobic peptide(e.g., consisting primarily of Tyr, Trp or Phe). The peptide moiety canbe a dendrimer peptide, constrained peptide or crosslinked peptide. Inanother alternative, the peptide moiety can include a hydrophobicmembrane translocation sequence (MTS). An exemplary hydrophobicMTS-containing peptide is RFGF having the amino acid sequenceAAVALLPAVLLALLAP (SEQ ID NO: 1831). An RFGF analogue (e.g., amino acidsequence AALLPVLLAAP (SEQ ID NO: 1832) containing a hydrophobic MTS canalso be a targeting moiety. The peptide moiety can be a “delivery”peptide, which can carry large polar molecules including peptides,oligonucleotides, and protein across cell membranes. For example,sequences from the HIV Tat protein (GRKKRRQRRRPPQ (SEQ ID NO: 1833) andthe Drosophila Antennapedia protein (RQIKIWFQNRRMKWKK (SEQ ID NO: 1834)have been found to be capable of functioning as delivery peptides. Apeptide or peptidomimetic can be encoded by a random sequence of DNA,such as a peptide identified from a phage-display library, orone-bead-one-compound (OBOC) combinatorial library (Lam et al., Nature,354:82-84, 1991). Examples of a peptide or peptidomimetic tethered to adsRNA agent via an incorporated monomer unit for cell targeting purposesis an arginine-glycine-aspartic acid (RGD)-peptide, or RGD mimic. Apeptide moiety can range in length from about 5 amino acids to about 40amino acids. The peptide moieties can have a structural modification,such as to increase stability or direct conformational properties. Anyof the structural modifications described below can be utilized.

An RGD peptide for use in the compositions and methods of the inventionmay be linear or cyclic, and may be modified, e.g., glycosylated ormethylated, to facilitate targeting to a specific tissue(s).RGD-containing peptides and peptidiomimemtics may include D-amino acids,as well as synthetic RGD mimics. In addition to RGD, one can use othermoieties that target the integrin ligand. Preferred conjugates of thisligand target PECAM-1 or VEGF.

A “cell permeation peptide” is capable of permeating a cell, e.g., amicrobial cell, such as a bacterial or fungal cell, or a mammalian cell,such as a human cell. A microbial cell-permeating peptide can be, forexample, an α-helical linear peptide (e.g., LL-37 or Ceropin P1), adisulfide bond-containing peptide (e.g., α-defensin, β-defensin orbactenecin), or a peptide containing only one or two dominating aminoacids (e.g., PR-39 or indolicidin). A cell permeation peptide can alsoinclude a nuclear localization signal (NLS). For example, a cellpermeation peptide can be a bipartite amphipathic peptide, such as MPG,which is derived from the fusion peptide domain of HIV-1 gp41 and theNLS of SV40 large T antigen (Simeoni et al., Nucl. Acids Res.31:2717-2724, 2003).

C. Carbohydrate Conjugates

In some embodiments of the compositions and methods of the invention, aniRNA oligonucleotide further comprises a carbohydrate. The carbohydrateconjugated iRNA are advantageous for the in vivo delivery of nucleicacids, as well as compositions suitable for in vivo therapeutic use, asdescribed herein. As used herein, “carbohydrate” refers to a compoundwhich is either a carbohydrate per se made up of one or moremonosaccharide units having at least 6 carbon atoms (which can belinear, branched or cyclic) with an oxygen, nitrogen or sulfur atombonded to each carbon atom; or a compound having as a part thereof acarbohydrate moiety made up of one or more monosaccharide units eachhaving at least six carbon atoms (which can be linear, branched orcyclic), with an oxygen, nitrogen or sulfur atom bonded to each carbonatom. Representative carbohydrates include the sugars (mono-, di-, tri-and oligosaccharides containing from about 4, 5, 6, 7, 8, or 9monosaccharide units), and polysaccharides such as starches, glycogen,cellulose and polysaccharide gums. Specific monosaccharides include HDVand above (e.g., C6, C7, or C8) sugars; di- and trisaccharides includesugars having two or three monosaccharide units (e.g., C6, C7, or C8).

In one embodiment, a carbohydrate conjugate for use in the compositionsand methods of the invention is a monosaccharide. In one embodiment, themonosaccharide is an N-acetylgalactosamine, such as

In another embodiment, a carbohydrate conjugate for use in thecompositions and methods of the invention is selected from the groupconsisting of:

Another representative carbohydrate conjugate for use in the embodimentsdescribed herein includes, but is not limited to,

-   -   (Formula XXIII), when one of X or Y is an oligonucleotide, the        other is a hydrogen.

In some embodiments, the carbohydrate conjugate further comprises one ormore additional ligands as described above, such as, but not limited to,a PK modulator and/or a cell permeation peptide.

D. Linkers

In some embodiments, the conjugate or ligand described herein can beattached to an iRNA oligonucleotide with various linkers that can becleavable or non-cleavable.

The term “linker” or “linking group” means an organic moiety thatconnects two parts of a compound, e.g., covalently attaches two parts ofa compound. Linkers typically comprise a direct bond or an atom such asoxygen or sulfur, a unit such as NR8, C(O), C(O)NH, SO, SO₂, SO₂NH or achain of atoms, such as, but not limited to, substituted orunsubstituted alkyl, substituted or unsubstituted alkenyl, substitutedor unsubstituted alkynyl, arylalkyl, arylalkenyl, arylalkynyl,heteroarylalkyl, heteroarylalkenyl, heteroarylalkynyl,heterocyclylalkyl, heterocyclylalkenyl, heterocyclylalkynyl, aryl,heteroaryl, heterocyclyl, cycloalkyl, cycloalkenyl, alkylarylalkyl,alkylarylalkenyl, alkylarylalkynyl, alkenylarylalkyl,alkenylarylalkenyl, alkenylarylalkynyl, alkynylarylalkyl,alkynylarylalkenyl, alkynylarylalkynyl, alkylheteroarylalkyl,alkylheteroarylalkenyl, alkylheteroarylalkynyl, alkenylheteroarylalkyl,alkenylheteroarylalkenyl, alkenylheteroarylalkynyl,alkynylheteroarylalkyl, alkynylheteroarylalkenyl,alkynylheteroarylalkynyl, alkylheterocyclylalkyl,alkylheterocyclylalkenyl, alkylhererocyclylalkynyl,alkenylheterocyclylalkyl, alkenylheterocyclylalkenyl,alkenylheterocyclylalkynyl, alkynylheterocyclylalkyl,alkynylheterocyclylalkenyl, alkynylheterocyclylalkynyl, alkylaryl,alkenylaryl, alkynylaryl, alkylheteroaryl, alkenylheteroaryl,alkynylhereroaryl, which one or more methylenes can be interrupted orterminated by O, S, S(O), SO₂, N(R8), C(O), substituted or unsubstitutedaryl, substituted or unsubstituted heteroaryl, substituted orunsubstituted heterocyclic; where R8 is hydrogen, acyl, aliphatic orsubstituted aliphatic. In one embodiment, the linker is between about1-24 atoms, 2-24, 3-24, 4-24, 5-24, 6-24, 6-18, 7-18, 8-18 atoms, 7-17,8-17, 6-16, 7-16, or 8-16 atoms.

A cleavable linking group is one which is sufficiently stable outsidethe cell, but which upon entry into a target cell is cleaved to releasethe two parts the linker is holding together. In a preferred embodiment,the cleavable linking group is cleaved at least about 10 times, 20,times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90times or more, or at least about 100 times faster in a target cell orunder a first reference condition (which can, e.g., be selected to mimicor represent intracellular conditions) than in the blood of a subject,or under a second reference condition (which can, e.g., be selected tomimic or represent conditions found in the blood or serum).

Cleavable linking groups are susceptible to cleavage agents, e.g., pH,redox potential or the presence of degradative molecules. Generally,cleavage agents are more prevalent or found at higher levels oractivities inside cells than in serum or blood. Examples of suchdegradative agents include: redox agents which are selected forparticular substrates or which have no substrate specificity, including,e.g., oxidative or reductive enzymes or reductive agents such asmercaptans, present in cells, that can degrade a redox cleavable linkinggroup by reduction; esterases; endosomes or agents that can create anacidic environment, e.g., those that result in a pH of five or lower;enzymes that can hydrolyze or degrade an acid cleavable linking group byacting as a general acid, peptidases (which can be substrate specific),and phosphatases.

A cleavable linkage group, such as a disulfide bond can be susceptibleto pH. The pH of human serum is 7.4, while the average intracellular pHis slightly lower, ranging from about 7.1-7.3. Endosomes have a moreacidic pH, in the range of 5.5-6.0, and lysosomes have an even moreacidic pH at around 5.0. Some linkers will have a cleavable linkinggroup that is cleaved at a preferred pH, thereby releasing a cationiclipid from the ligand inside the cell, or into the desired compartmentof the cell.

A linker can include a cleavable linking group that is cleavable by aparticular enzyme. The type of cleavable linking group incorporated intoa linker can depend on the cell to be targeted. For example, aliver-targeting ligand can be linked to a cationic lipid through alinker that includes an ester group. Liver cells are rich in esterases,and therefore the linker will be cleaved more efficiently in liver cellsthan in cell types that are not esterase-rich. Other cell-types rich inesterases include cells of the lung, renal cortex, and testis.

Linkers that contain peptide bonds can be used when targeting cell typesrich in peptidases, such as liver cells and synoviocytes.

In general, the suitability of a candidate cleavable linking group canbe evaluated by testing the ability of a degradative agent (orcondition) to cleave the candidate linking group. It will also bedesirable to also test the candidate cleavable linking group for theability to resist cleavage in the blood or when in contact with othernon-target tissue. Thus, one can determine the relative susceptibilityto cleavage between a first and a second condition, where the first isselected to be indicative of cleavage in a target cell and the second isselected to be indicative of cleavage in other tissues or biologicalfluids, e.g., blood or serum. The evaluations can be carried out in cellfree systems, in cells, in cell culture, in organ or tissue culture, orin whole animals. It can be useful to make initial evaluations incell-free or culture conditions and to confirm by further evaluations inwhole animals. In preferred embodiments, useful candidate compounds arecleaved at least about 2, 4, 10, 20, 30, 40, 50, 60, 70, 80, 90, orabout 100 times faster in the cell (or under in vitro conditionsselected to mimic intracellular conditions) as compared to blood orserum (or under in vitro conditions selected to mimic extracellularconditions).

i. Redox Cleavable Linking Groups

In one embodiment, a cleavable linking group is a redox cleavablelinking group that is cleaved upon reduction or oxidation. An example ofreductively cleavable linking group is a disulphide linking group(—S—S—). To determine if a candidate cleavable linking group is asuitable “reductively cleavable linking group,” or for example issuitable for use with a particular iRNA moiety and particular targetingagent one can look to methods described herein. For example, a candidatecan be evaluated by incubation with dithiothreitol (DTT), or otherreducing agent using reagents know in the art, which mimic the rate ofcleavage which would be observed in a cell, e.g., a target cell. Thecandidates can also be evaluated under conditions which are selected tomimic blood or serum conditions. In one, candidate compounds are cleavedby at most about 10% in the blood. In other embodiments, usefulcandidate compounds are degraded at least about 2, 4, 10, 20, 30, 40,50, 60, 70, 80, 90, or about 100 times faster in the cell (or under invitro conditions selected to mimic intracellular conditions) as comparedto blood (or under in vitro conditions selected to mimic extracellularconditions). The rate of cleavage of candidate compounds can bedetermined using standard enzyme kinetics assays under conditions chosento mimic intracellular media and compared to conditions chosen to mimicextracellular media.

ii. Phosphate-Based Cleavable Linking Groups

In another embodiment, a cleavable linker comprises a phosphate-basedcleavable linking group. A phosphate-based cleavable linking group iscleaved by agents that degrade or hydrolyze the phosphate group. Anexample of an agent that cleaves phosphate groups in cells are enzymessuch as phosphatases in cells. Examples of phosphate-based linkinggroups are —O—P(O)(ORk)-O—, —O—P(S)(ORk)-O—, —O—P(S)(SRk)-O—,—S—P(O)(ORk)-O—, —O—P(O)(ORk)-S—, —S—P(O)(ORk)-S—, —O—P(S)(ORk)-S—,—S—P(S)(ORk)-O—, —O—P(O)(Rk)-O—, —O—P(S)(Rk)-O—, —S—P(O)(Rk)-O—,—S—P(S)(Rk)-O—, —S—P(O)(Rk)-S—, —O—P(S)(Rk)-S—. Preferred embodimentsare —O—P(O)(OH)—O—, —O—P(S)(OH)—O—, —O—P(S)(SH)—O—, —S—P(O)(OH)—O—,—O—P(O)(OH)—S—, —S—P(O)(OH)—S—, —O—P(S)(OH)—S—, —S—P(S)(OH)—O—,—O—P(O)(H)—O—, —O—P(S)(H)—O—, —S—P(O)(H)—O, —S—P(S)(H)—O—,—S—P(O)(H)—S—, —O—P(S)(H)—S—. A preferred embodiment is —O—P(O)(OH)—O—.These candidates can be evaluated using methods analogous to thosedescribed above.

iii. Acid Cleavable Linking Groups

In another embodiment, a cleavable linker comprises an acid cleavablelinking group. An acid cleavable linking group is a linking group thatis cleaved under acidic conditions. In preferred embodiments acidcleavable linking groups are cleaved in an acidic environment with a pHof about 6.5 or lower (e.g., about 6.0, 5.75, 5.5, 5.25, 5.0, or lower),or by agents such as enzymes that can act as a general acid. In a cell,specific low pH organelles, such as endosomes and lysosomes can providea cleaving environment for acid cleavable linking groups. Examples ofacid cleavable linking groups include but are not limited to hydrazones,esters, and esters of amino acids. Acid cleavable groups can have thegeneral formula —C═NN—, C(O)O, or —OC(O). A preferred embodiment is whenthe carbon attached to the oxygen of the ester (the alkoxy group) is anaryl group, substituted alkyl group, or tertiary alkyl group such asdimethyl pentyl or t-butyl. These candidates can be evaluated usingmethods analogous to those described above.

iv. Ester-Based Linking Groups

In another embodiment, a cleavable linker comprises an ester-basedcleavable linking group. An ester-based cleavable linking group iscleaved by enzymes such as esterases and amidases in cells. Examples ofester-based cleavable linking groups include but are not limited toesters of alkylene, alkenylene and alkynylene groups. Ester cleavablelinking groups have the general formula —C(O)O—, or —OC(O)—. Thesecandidates can be evaluated using methods analogous to those describedabove.

v. Peptide-Based Cleaving Groups

In yet another embodiment, a cleavable linker comprises a peptide-basedcleavable linking group. A peptide-based cleavable linking group iscleaved by enzymes such as peptidases and proteases in cells.Peptide-based cleavable linking groups are peptide bonds formed betweenamino acids to yield oligopeptides (e.g., dipeptides, tripeptides etc.)and polypeptides. Peptide-based cleavable groups do not include theamide group (—C(O)NH—). The amide group can be formed between anyalkylene, alkenylene or alkynelene. A peptide bond is a special type ofamide bond formed between amino acids to yield peptides and proteins.The peptide based cleavage group is generally limited to the peptidebond (i.e., the amide bond) formed between amino acids yielding peptidesand proteins and does not include the entire amide functional group.Peptide-based cleavable linking groups have the general formula—NHCHRAC(O)NHCHRBC(O)—, where RA and RB are the R groups of the twoadjacent amino acids. These candidates can be evaluated using methodsanalogous to those described above.

In one embodiment, an iRNA of the invention is conjugated to acarbohydrate through a linker. Non-limiting examples of iRNAcarbohydrate conjugates with linkers of the compositions and methods ofthe invention include, but are not limited to,

when one of X or Y is an oligonucleotide, the other is a hydrogen.

In certain embodiments of the compositions and methods of the invention,a ligand is one or more “GalNAc” (N-acetylgalactosamine) derivativesattached through a bivalent or trivalent branched linker.

In one embodiment, a dsRNA of the invention is conjugated to a bivalentor trivalent branched linker selected from the group of structures shownin any of formula (XXXII)-(XXXV):

wherein:q2A, q2B, q3A, q3B, q4A, q4B, q5A, q5B and q5C represent independentlyfor each occurrence 0-20 and wherein the repeating unit can be the sameor different;P^(2A), P^(2B), P^(3A), P^(3B), P^(4A), P^(4B), P^(5A), P^(5B), P^(5C),T^(2A), T^(2B), T^(3A), T^(3B), T^(4A), T^(4B), T^(4A), T^(5B), T^(5C)are each independently for each occurrence absent, CO, NH, O, S, OC(O),NHC(O), CH₂, CH₂NH or CH₂O;Q^(2A), Q^(2B), Q^(3A), Q^(3B), Q^(4A), Q^(4B), Q^(5A), Q^(5B), Q^(5C)are independently for each occurrence absent, alkylene, substitutedalkylene wherein one or more methylenes can be interrupted or terminatedby one or more of O, S, S(O), SO₂, N(R^(N)), C(R′)═C(R″), C≡C or C(O);R^(2A), R^(2B), R^(3A), R^(3B), R^(4A), R^(4B), R^(5A), R^(5B), R^(5C)are each independently for each occurrence absent, NH, O, S, CH₂, C(O)O,C(O)NH, NHCH(R^(a))C(O), —C(O)—CH(R^(a))—NH—, CO, CH═N—O,

or heterocyclyl;

L^(2A), L^(2B), L^(3A), L^(3B), L^(4A), L^(4B), L^(5A), L^(5B) andL^(5C) represent the ligand; i.e. each independently for each occurrencea monosaccharide (such as GalNAc), disaccharide, trisaccharide,tetrasaccharide, oligosaccharide, or polysaccharide; and R^(a) is H oramino acid side chain. Trivalent conjugating GalNAc derivatives areparticularly useful for use with RNAi agents for inhibiting theexpression of a target gene, such as those of formula (XXXV):

-   -   wherein L^(5A), L^(5B) and L^(5C) represent a monosaccharide,        such as GalNAc derivative.

Examples of suitable bivalent and trivalent branched linker groupsconjugating GalNAc derivatives include, but are not limited to, thestructures recited above as formulas II, VII, XI, X, and XIII.

Representative U.S. patents that teach the preparation of RNA conjugatesinclude, but are not limited to, U.S. Pat. Nos. 4,828,979; 4,948,882;5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717,5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077;5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735;4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,835,263; 4,876,335;4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,136; 5,082,830;5,112,963; 5,214,136; 5,245,022; 5,254,469; 5,258,506; 5,262,536;5,272,250; 5,292,873; 5,317,098; 5,371,241, 5,391,723; 5,416,203,5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552; 5,567,810;5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923;5,599,928 and 5,688,941; 6,294,664; 6,320,017; 6,576,752; 6,783,931;6,900,297; 7,037,646; 8,106,022, the entire contents of each of whichare hereby incorporated herein by reference.

It is not necessary for all positions in a given compound to beuniformly modified, and in fact more than one of the aforementionedmodifications can be incorporated in a single compound or even at asingle nucleoside within an iRNA. The present invention also includesiRNA compounds that are chimeric compounds.

“Chimeric” iRNA compounds or “chimeras,” in the context of thisinvention, are iRNA compounds, preferably dsRNAs, which contain two ormore chemically distinct regions, each made up of at least one monomerunit, i.e., a nucleotide in the case of a dsRNA compound. These iRNAstypically contain at least one region wherein the RNA is modified so asto confer upon the iRNA increased resistance to nuclease degradation,increased cellular uptake, and/or increased binding affinity for thetarget nucleic acid. An additional region of the iRNA can serve as asubstrate for enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. Byway of example, RNase H is a cellular endonuclease which cleaves the RNAstrand of an RNA:DNA duplex. Activation of RNase H, therefore, resultsin cleavage of the RNA target, thereby greatly enhancing the efficiencyof iRNA inhibition of gene expression. Consequently, comparable resultscan often be obtained with shorter iRNAs when chimeric dsRNAs are used,compared to phosphorothioate deoxy dsRNAs hybridizing to the same targetregion. Cleavage of the RNA target can be routinely detected by gelelectrophoresis and, if necessary, associated nucleic acid hybridizationtechniques known in the art.

In certain instances, the RNA of an iRNA can be modified by a non-ligandgroup. A number of non-ligand molecules have been conjugated to iRNAs inorder to enhance the activity, cellular distribution or cellular uptakeof the iRNA, and procedures for performing such conjugations areavailable in the scientific literature. Such non-ligand moieties haveincluded lipid moieties, such as cholesterol (Kubo, T. et al., Biochem.Biophys. Res. Comm., 2007, 365(1):54-61; Letsinger et al., Proc. Natl.Acad. Sci. USA, 1989, 86:6553), cholic acid (Manoharan et al., Bioorg.Med. Chem. Lett., 1994, 4:1053), a thioether, e.g., hexyl-S-tritylthiol(Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660:306; Manoharan etal., Bioorg. Med. Chem. Let., 1993, 3:2765), a thiocholesterol(Oberhauser et al., Nucl. Acids Res., 1992, 20:533), an aliphatic chain,e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al., EMBO J.,1991, 10:111; Kabanov et al., FEBS Lett., 1990, 259:327; Svinarchuk etal., Biochimie, 1993, 75:49), a phospholipid, e.g.,di-hexadecyl-rac-glycerol or triethylammonium1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al.,Tetrahedron Lett., 1995, 36:3651; Shea et al., Nucl. Acids Res., 1990,18:3777), a polyamine or a polyethylene glycol chain (Manoharan et al.,Nucleosides & Nucleotides, 1995, 14:969), or adamantane acetic acid(Manoharan et al., Tetrahedron Lett., 1995, 36:3651), a palmityl moiety(Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229), or anoctadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke etal., J. Pharmacol. Exp. Ther., 1996, 277:923). Representative UnitedStates patents that teach the preparation of such RNA conjugates havebeen listed above. Typical conjugation protocols involve the synthesisof an RNAs bearing an aminolinker at one or more positions of thesequence. The amino group is then reacted with the molecule beingconjugated using appropriate coupling or activating reagents. Theconjugation reaction can be performed either with the RNA still bound tothe solid support or following cleavage of the RNA, in solution phase.Purification of the RNA conjugate by HPLC typically affords the pureconjugate.

V. Delivery of an iRNA of the Invention

The delivery of an iRNA of the invention to a cell e.g., a cell within asubject, such as a human subject (e.g., a subject in need thereof, suchas a subject having a disease, disorder or condition associated with HDVinfection) can be achieved in a number of different ways. For example,delivery may be performed by contacting a cell with an iRNA of theinvention either in vitro or in vivo. In vivo delivery may also beperformed directly by administering a composition comprising an iRNA,e.g., a dsRNA, to a subject. Alternatively, in vivo delivery may beperformed indirectly by administering one or more vectors that encodeand direct the expression of the iRNA. These alternatives are discussedfurther below.

In general, any method of delivering a nucleic acid molecule (in vitroor in vivo) can be adapted for use with an iRNA of the invention (seee.g., Akhtar S. and Julian R L. (1992) Trends Cell. Biol. 2(5):139-144and WO94/02595, which are incorporated herein by reference in theirentireties). For in vivo delivery, factors to consider in order todeliver an iRNA molecule include, for example, biological stability ofthe delivered molecule, prevention of non-specific effects, andaccumulation of the delivered molecule in the target tissue. Thenon-specific effects of an iRNA can be minimized by localadministration, for example, by direct injection or implantation into atissue or topically administering the preparation. Local administrationto a treatment site maximizes local concentration of the agent, limitsthe exposure of the agent to systemic tissues that can otherwise beharmed by the agent or that can degrade the agent, and permits a lowertotal dose of the iRNA molecule to be administered. Several studies haveshown successful knockdown of gene products when an iRNA is administeredlocally. For example, intraocular delivery of a VEGF dsRNA byintravitreal injection in cynomolgus monkeys (Tolentino, M J., et al(2004) Retina 24:132-138) and subretinal injections in mice (Reich, SJ., et al (2003) Mol. Vis. 9:210-216) were both shown to preventneovascularization in an experimental model of age-related maculardegeneration. In addition, direct intratumoral injection of a dsRNA inmice reduces tumor volume (Pille, J., et al (2005) Mol. Ther.11:267-274) and can prolong survival of tumor-bearing mice (Kim, W J.,et al (2006) Mol. Ther. 14:343-350; Li, S., et al (2007) Mol. Ther.15:515-523). RNA interference has also shown success with local deliveryto the CNS by direct injection (Dorn, G., et al. (2004) Nucleic Acids32:e49; Tan, P H., et al (2005) Gene Ther. 12:59-66; Makimura, H., et al(2002) BMC Neurosci. 3:18; Shishkina, G T., et al (2004) Neuroscience129:521-528; Thakker, E R., et al (2004) Proc. Natl. Acad. Sci. U.S.A.101:17270-17275; Akaneya, Y., et al (2005) J. Neurophysiol. 93:594-602)and to the lungs by intranasal administration (Howard, K A., et al(2006) Mol. Ther. 14:476-484; Zhang, X., et al (2004) J. Biol. Chem.279:10677-10684; Bitko, V., et al (2005) Nat. Med. 11:50-55). Foradministering an iRNA systemically for the treatment of a disease, theRNA can be modified or alternatively delivered using a drug deliverysystem; both methods act to prevent the rapid degradation of the dsRNAby endo- and exo-nucleases in vivo. Modification of the RNA or thepharmaceutical carrier can also permit targeting of the iRNA compositionto the target tissue and avoid undesirable off-target effects. iRNAmolecules can be modified by chemical conjugation to lipophilic groupssuch as cholesterol to enhance cellular uptake and prevent degradation.For example, an iRNA directed against ApoB conjugated to a lipophiliccholesterol moiety was injected systemically into mice and resulted inknockdown of apoB mRNA in both the liver and jejunum (Soutschek, J., etal (2004) Nature 432:173-178). Conjugation of an iRNA to an aptamer hasbeen shown to inhibit tumor growth and mediate tumor regression in amouse model of prostate cancer (McNamara, J O., et al (2006) Nat.Biotechnol. 24:1005-1015). In an alternative embodiment, the iRNA can bedelivered using drug delivery systems such as a nanoparticle, adendrimer, a polymer, liposomes, or a cationic delivery system.Positively charged cationic delivery systems facilitate binding of aniRNA molecule (negatively charged) and also enhance interactions at thenegatively charged cell membrane to permit efficient uptake of an iRNAby the cell. Cationic lipids, dendrimers, or polymers can either bebound to an iRNA, or induced to form a vesicle or micelle (see e.g., KimS H., et al (2008) Journal of Controlled Release 129(2):107-116) thatencases an iRNA. The formation of vesicles or micelles further preventsdegradation of the iRNA when administered systemically. Methods formaking and administering cationic-iRNA complexes are well within theabilities of one skilled in the art (see e.g., Sorensen, D R., et al(2003) J. Mol. Biol 327:761-766; Verma, U N., et al (2003) Clin. CancerRes. 9:1291-1300; Arnold, A S et al (2007) J. Hypertens. 25:197-205,which are incorporated herein by reference in their entirety). Somenon-limiting examples of drug delivery systems useful for systemicdelivery of iRNAs include DOTAP (Sorensen, D R., et al (2003), supra;Verma, U N., et al (2003), supra), Oligofectamine, “solid nucleic acidlipid particles” (Zimmermann, T S., et al (2006) Nature 441:111-114),cardiolipin (Chien, P Y., et al (2005) Cancer Gene Ther. 12:321-328;Pal, A., et al (2005) Int J. Oncol. 26:1087-1091), polyethyleneimine(Bonnet M E., et al (2008) Pharm. Res. August 16 Epub ahead of print;Aigner, A. (2006) J. Biomed. Biotechnol. 71659), Arg-Gly-Asp (RGD)peptides (Liu, S. (2006) Mol. Pharm. 3:472-487), and polyamidoamines(Tomalia, D A., et al (2007) Biochem. Soc. Trans. 35:61-67; Yoo, H., etal (1999) Pharm. Res. 16:1799-1804). In some embodiments, an iRNA formsa complex with cyclodextrin for systemic administration. Methods foradministration and pharmaceutical compositions of iRNAs andcyclodextrins can be found in U.S. Pat. No. 7,427,605, which is hereinincorporated by reference in its entirety.

A. Vector Encoded iRNAs of the Invention

iRNA targeting the HDV gene can be expressed from transcription unitsinserted into DNA or RNA vectors (see, e.g., Couture, A, et al., TIG.(1996), 12:5-10; Skillern, A., et al., International PCT Publication No.WO 00/22113, Conrad, International PCT Publication No. WO 00/22114, andConrad, U.S. Pat. No. 6,054,299). Expression can be transient (on theorder of hours to weeks) or sustained (weeks to months or longer),depending upon the specific construct used and the target tissue or celltype. These transgenes can be introduced as a linear construct, acircular plasmid, or a viral vector, which can be an integrating ornon-integrating vector. The transgene can also be constructed to permitit to be inherited as an extrachromosomal plasmid (Gassmann, et al.,Proc. Natl. Acad. Sci. USA (1995) 92:1292).

The individual strand or strands of an iRNA can be transcribed from apromoter on an expression vector. Where two separate strands are to beexpressed to generate, for example, a dsRNA, two separate expressionvectors can be co-introduced (e.g., by transfection or infection) into atarget cell. Alternatively each individual strand of a dsRNA can betranscribed by promoters both of which are located on the sameexpression plasmid. In one embodiment, a dsRNA is expressed as invertedrepeat polynucleotides joined by a linker polynucleotide sequence suchthat the dsRNA has a stem and loop structure.

iRNA expression vectors are generally DNA plasmids or viral vectors.Expression vectors compatible with eukaryotic cells, preferably thosecompatible with vertebrate cells, can be used to produce recombinantconstructs for the expression of an iRNA as described herein. Eukaryoticcell expression vectors are well known in the art and are available froma number of commercial sources. Typically, such vectors are providedcontaining convenient restriction sites for insertion of the desirednucleic acid segment. Delivery of iRNA expressing vectors can besystemic, such as by intravenous or intramuscular administration, byadministration to target cells ex-planted from the patient followed byreintroduction into the patient, or by any other means that allows forintroduction into a desired target cell.

iRNA expression plasmids can be transfected into target cells as acomplex with cationic lipid carriers (e.g., Oligofectamine) ornon-cationic lipid-based carriers (e.g., Transit-TKO™). Multiple lipidtransfections for iRNA-mediated knockdowns targeting different regionsof a target RNA over a period of a week or more are also contemplated bythe invention. Successful introduction of vectors into host cells can bemonitored using various known methods. For example, transienttransfection can be signaled with a reporter, such as a fluorescentmarker, such as Green Fluorescent Protein (GFP). Stable transfection ofcells ex vivo can be ensured using markers that provide the transfectedcell with resistance to specific environmental factors (e.g.,antibiotics and drugs), such as hygromycin B resistance.

Viral vector systems which can be utilized with the methods andcompositions described herein include, but are not limited to, (a)adenovirus vectors; (b) retrovirus vectors, including but not limited tolentiviral vectors, moloney murine leukemia virus, etc.; (c)adeno-associated virus vectors; (d) herpes simplex virus vectors; (e) SV40 vectors; (f) polyoma virus vectors; (g) papilloma virus vectors; (h)picornavirus vectors; (i) pox virus vectors such as an orthopox, e.g.,vaccinia virus vectors or avipox, e.g. canary pox or fowl pox; and (j) ahelper-dependent or gutless adenovirus. Replication-defective virusescan also be advantageous. Different vectors will or will not becomeincorporated into the cells' genome. The constructs can include viralsequences for transfection, if desired. Alternatively, the construct canbe incorporated into vectors capable of episomal replication, e.g. EPVand EBV vectors. Constructs for the recombinant expression of an iRNAwill generally require regulatory elements, e.g., promoters, enhancers,etc., to ensure the expression of the iRNA in target cells. Otheraspects to consider for vectors and constructs are further describedbelow.

Vectors useful for the delivery of an iRNA will include regulatoryelements (promoter, enhancer, etc.) sufficient for expression of theiRNA in the desired target cell or tissue. The regulatory elements canbe chosen to provide either constitutive or regulated/inducibleexpression.

Expression of the iRNA can be precisely regulated, for example, by usingan inducible regulatory sequence that is sensitive to certainphysiological regulators, e.g., circulating glucose levels, or hormones(Docherty et al., 1994, FASEB J. 8:20-24). Such inducible expressionsystems, suitable for the control of dsRNA expression in cells or inmammals include, for example, regulation by ecdysone, by estrogen,progesterone, tetracycline, chemical inducers of dimerization, andisopropyl-beta-D1-thiogalactopyranoside (IPTG). A person skilled in theart would be able to choose the appropriate regulatory/promoter sequencebased on the intended use of the iRNA transgene.

Viral vectors that contain nucleic acid sequences encoding an iRNA canbe used. For example, a retroviral vector can be used (see Miller etal., Meth. Enzymol. 217:581-599 (1993)). These retroviral vectorscontain the components necessary for the correct packaging of the viralgenome and integration into the host cell DNA. The nucleic acidsequences encoding an iRNA are cloned into one or more vectors, whichfacilitate delivery of the nucleic acid into a patient. More detailabout retroviral vectors can be found, for example, in Boesen et al.,Biotherapy 6:291-302 (1994), which describes the use of a retroviralvector to deliver the mdr1 gene to hematopoietic stem cells in order tomake the stem cells more resistant to chemotherapy. Other referencesillustrating the use of retroviral vectors in gene therapy are: Cloweset al., J. Clin. Invest. 93:644-651 (1994); Kiem et al., Blood83:1467-1473 (1994); Salmons and Gunzberg, Human Gene Therapy 4:129-141(1993); and Grossman and Wilson, Curr. Opin. in Genetics and Devel.3:110-114 (1993). Lentiviral vectors contemplated for use include, forexample, the HIV based vectors described in U.S. Pat. Nos. 6,143,520;5,665,557; and 5,981,276, which are herein incorporated by reference.

Adenoviruses are also contemplated for use in delivery of iRNAs of theinvention. Adenoviruses are especially attractive vehicles, e.g., fordelivering genes to respiratory epithelia. Adenoviruses naturally infectrespiratory epithelia where they cause a mild disease. Other targets foradenovirus-based delivery systems are liver, the central nervous system,endothelial cells, and muscle. Adenoviruses have the advantage of beingcapable of infecting non-dividing cells. Kozarsky and Wilson, CurrentOpinion in Genetics and Development 3:499-503 (1993) present a review ofadenovirus-based gene therapy. Bout et al., Human Gene Therapy 5:3-10(1994) demonstrated the use of adenovirus vectors to transfer genes tothe respiratory epithelia of rhesus monkeys. Other instances of the useof adenoviruses in gene therapy can be found in Rosenfeld et al.,Science 252:431-434 (1991); Rosenfeld et al., Cell 68:143-155 (1992);Mastrangeli et al., J. Clin. Invest. 91:225-234 (1993); PCT PublicationWO94/12649; and Wang, et al., Gene Therapy 2:775-783 (1995). A suitableAV vector for expressing an iRNA featured in the invention, a method forconstructing the recombinant AV vector, and a method for delivering thevector into target cells, are described in Xia H et al. (2002), Nat.Biotech. 20: 1006-1010.

Adeno-associated virus (AAV) vectors may also be used to delivery aniRNA of the invention (Walsh et al., Proc. Soc. Exp. Biol. Med.204:289-300 (1993); U.S. Pat. No. 5,436,146). In one embodiment, theiRNA can be expressed as two separate, complementary single-stranded RNAmolecules from a recombinant AAV vector having, for example, either theU6 or H1 RNA promoters, or the cytomegalovirus (CMV) promoter. SuitableAAV vectors for expressing the dsRNA featured in the invention, methodsfor constructing the recombinant AV vector, and methods for deliveringthe vectors into target cells are described in Samulski R et al. (1987),J. Virol. 61: 3096-3101; Fisher K J et al. (1996), J. Virol, 70:520-532; Samulski R et al. (1989), J. Virol. 63: 3822-3826; U.S. Pat.Nos. 5,252,479; 5,139,941; International Patent Application No. WO94/13788; and International Patent Application No. WO 93/24641, theentire disclosures of which are herein incorporated by reference.

Another viral vector suitable for delivery of an iRNA of the inventionis a pox virus such as a vaccinia virus, for example an attenuatedvaccinia such as Modified Virus Ankara (MVA) or NYVAC, an avipox such asfowl pox or canary pox.

The tropism of viral vectors can be modified by pseudotyping the vectorswith envelope proteins or other surface antigens from other viruses, orby substituting different viral capsid proteins, as appropriate. Forexample, lentiviral vectors can be pseudotyped with surface proteinsfrom vesicular stomatitis virus (VSV), rabies, Ebola, Mokola, and thelike. AAV vectors can be made to target different cells by engineeringthe vectors to express different capsid protein serotypes; see, e.g.,Rabinowitz J E et al. (2002), J Virol 76:791-801, the entire disclosureof which is herein incorporated by reference.

The pharmaceutical preparation of a vector can include the vector in anacceptable diluent, or can include a slow release matrix in which thegene delivery vehicle is imbedded. Alternatively, where the completegene delivery vector can be produced intact from recombinant cells,e.g., retroviral vectors, the pharmaceutical preparation can include oneor more cells which produce the gene delivery system.

VI. Pharmaceutical Compositions of the Invention

The present invention also includes pharmaceutical compositions andformulations which include the iRNAs of the invention. In oneembodiment, provided herein are pharmaceutical compositions containingan iRNA, as described herein, and a pharmaceutically acceptable carrier.The pharmaceutical compositions containing the iRNA are useful fortreating a disease or disorder associated with the expression oractivity of an HDV gene. Such pharmaceutical compositions are formulatedbased on the mode of delivery. One example is compositions that areformulated for systemic administration via parenteral delivery, e.g., bysubcutaneous, intramuscularly (IM), (SC) or intravenous (IV) delivery.Another example is compositions that are formulated for direct deliveryinto the brain parenchyma, e.g., by infusion into the brain, such as bycontinuous pump infusion. The pharmaceutical compositions of theinvention may be administered in dosages sufficient to inhibitexpression of an HDV gene.

In one embodiment, an iRNA agent of the invention is administered to asubject as a weight-based dose. A “weight-based dose” (e.g., a dose inmg/kg) is a dose of the iRNA agent that will change depending on thesubject's weight. In another embodiment, an iRNA agent is administeredto a subject as a fixed dose. A “fixed dose” (e.g., a dose in mg) meansthat one dose of an iRNA agent is used for all subjects regardless ofany specific subject-related factors, such as weight. In one particularembodiment, a fixed dose of an iRNA agent of the invention is based on apredetermined weight or age.

In general, a suitable dose of an iRNA of the invention will be in therange of about 0.001 to about 200.0 milligrams per kilogram body weightof the recipient per day, generally in the range of about 1 to 50 mg perkilogram body weight per day. For example, the dsRNA can be administeredat about 0.01 mg/kg, about 0.05 mg/kg, about 0.5 mg/kg, about 1 mg/kg,about 1.5 mg/kg, about 2 mg/kg, about 3 mg/kg, about 10 mg/kg, about 20mg/kg, about 30 mg/kg, about 40 mg/kg, or about 50 mg/kg per singledose.

For example, the dsRNA may be administered at a dose of about 0.1, 0.2,0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7,1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2,3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7,4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2,6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7,7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2,9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or about 10 mg/kg. Values and rangesintermediate to the recited values are also intended to be part of thisinvention.

In another embodiment, the dsRNA is administered at a dose of about 0.1to about 50 mg/kg, about 0.25 to about 50 mg/kg, about 0.5 to about 50mg/kg, about 0.75 to about 50 mg/kg, about 1 to about 50 mg/kg, about1.5 to about 50 mg/kg, about 2 to about 50 mg/kg, about 2.5 to about 50mg/kg, about 3 to about 50 mg/kg, about 3.5 to about 50 mg/kg, about 4to about 50 mg/kg, about 4.5 to about 50 mg/kg, about 5 to about 50mg/kg, about 7.5 to about 50 mg/kg, about 10 to about 50 mg/kg, about 15to about 50 mg/kg, about 20 to about 50 mg/kg, about 20 to about 50mg/kg, about 25 to about 50 mg/kg, about 25 to about 50 mg/kg, about 30to about 50 mg/kg, about 35 to about 50 mg/kg, about 40 to about 50mg/kg, about 45 to about 50 mg/kg, about 0.1 to about 45 mg/kg, about0.25 to about 45 mg/kg, about 0.5 to about 45 mg/kg, about 0.75 to about45 mg/kg, about 1 to about 45 mg/kg, about 1.5 to about 45 mg/kg, about2 to about 45 mg/kg, about 2.5 to about 45 mg/kg, about 3 to about 45mg/kg, about 3.5 to about 45 mg/kg, about 4 to about 45 mg/kg, about 4.5to about 45 mg/kg, about 5 to about 45 mg/kg, about 7.5 to about 45mg/kg, about 10 to about 45 mg/kg, about 15 to about 45 mg/kg, about 20to about 45 mg/kg, about 20 to about 45 mg/kg, about 25 to about 45mg/kg, about 25 to about 45 mg/kg, about 30 to about 45 mg/kg, about 35to about 45 mg/kg, about 40 to about 45 mg/kg, about 0.1 to about 40mg/kg, about 0.25 to about 40 mg/kg, about 0.5 to about 40 mg/kg, about0.75 to about 40 mg/kg, about 1 to about 40 mg/kg, about 1.5 to about 40mg/kg, about 2 to about 40 mg/kg, about 2.5 to about 40 mg/kg, about 3to about 40 mg/kg, about 3.5 to about 40 mg/kg, about 4 to about 40mg/kg, about 4.5 to about 40 mg/kg, about 5 to about 40 mg/kg, about 7.5to about 40 mg/kg, about 10 to about 40 mg/kg, about 15 to about 40mg/kg, about 20 to about 40 mg/kg, about 20 to about 40 mg/kg, about 25to about 40 mg/kg, about 25 to about 40 mg/kg, about 30 to about 40mg/kg, about 35 to about 40 mg/kg, about 0.1 to about 30 mg/kg, about0.25 to about 30 mg/kg, about 0.5 to about 30 mg/kg, about 0.75 to about30 mg/kg, about 1 to about 30 mg/kg, about 1.5 to about 30 mg/kg, about2 to about 30 mg/kg, about 2.5 to about 30 mg/kg, about 3 to about 30mg/kg, about 3.5 to about 30 mg/kg, about 4 to about 30 mg/kg, about 4.5to about 30 mg/kg, about 5 to about 30 mg/kg, about 7.5 to about 30mg/kg, about 10 to about 30 mg/kg, about 15 to about 30 mg/kg, about 20to about 30 mg/kg, about 20 to about 30 mg/kg, about 25 to about 30mg/kg, about 0.1 to about 20 mg/kg, about 0.25 to about 20 mg/kg, about0.5 to about 20 mg/kg, about 0.75 to about 20 mg/kg, about 1 to about 20mg/kg, about 1.5 to about 20 mg/kg, about 2 to about 20 mg/kg, about 2.5to about 20 mg/kg, about 3 to about 20 mg/kg, about 3.5 to about 20mg/kg, about 4 to about 20 mg/kg, about 4.5 to about 20 mg/kg, about 5to about 20 mg/kg, about 7.5 to about 20 mg/kg, about 10 to about 20mg/kg, or about 15 to about 20 mg/kg. Values and ranges intermediate tothe recited values are also intended to be part of this invention.

For example, the dsRNA may be administered at a dose of about 0.01,0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5,0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2,2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5,3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5,5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5,6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8,8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5,9.6, 9.7, 9.8, 9.9, or about 10 mg/kg. Values and ranges intermediate tothe recited values are also intended to be part of this invention.

In another embodiment, the dsRNA is administered at a dose of about 0.5to about 50 mg/kg, about 0.75 to about 50 mg/kg, about 1 to about 50mg/kg, about 1.5 to about 50 mg/kg, about 2 to about 50 mg/kg, about 2.5to about 50 mg/kg, about 3 to about 50 mg/kg, about 3.5 to about 50mg/kg, about 4 to about 50 mg/kg, about 4.5 to about 50 mg/kg, about 5to about 50 mg/kg, about 7.5 to about 50 mg/kg, about 10 to about 50mg/kg, about 15 to about 50 mg/kg, about 20 to about 50 mg/kg, about 20to about 50 mg/kg, about 25 to about 50 mg/kg, about 25 to about 50mg/kg, about 30 to about 50 mg/kg, about 35 to about 50 mg/kg, about 40to about 50 mg/kg, about 45 to about 50 mg/kg, about 0.5 to about 45mg/kg, about 0.75 to about 45 mg/kg, about 1 to about 45 mg/kg, about1.5 to about 45 mg/kg, about 2 to about 45 mg/kg, about 2.5 to about 45mg/kg, about 3 to about 45 mg/kg, about 3.5 to about 45 mg/kg, about 4to about 45 mg/kg, about 4.5 to about 45 mg/kg, about 5 to about 45mg/kg, about 7.5 to about 45 mg/kg, about 10 to about 45 mg/kg, about 15to about 45 mg/kg, about 20 to about 45 mg/kg, about 20 to about 45mg/kg, about 25 to about 45 mg/kg, about 25 to about 45 mg/kg, about 30to about 45 mg/kg, about 35 to about 45 mg/kg, about 40 to about 45mg/kg, about 0.5 to about 40 mg/kg, about 0.75 to about 40 mg/kg, about1 to about 40 mg/kg, about 1.5 to about 40 mg/kg, about 2 to about 40mg/kg, about 2.5 to about 40 mg/kg, about 3 to about 40 mg/kg, about 3.5to about 40 mg/kg, about 4 to about 40 mg/kg, about 4.5 to about 40mg/kg, about 5 to about 40 mg/kg, about 7.5 to about 40 mg/kg, about 10to about 40 mg/kg, about 15 to about 40 mg/kg, about 20 to about 40mg/kg, about 20 to about 40 mg/kg, about 25 to about 40 mg/kg, about 25to about 40 mg/kg, about 30 to about 40 mg/kg, about 35 to about 40mg/kg, about 0.5 to about 30 mg/kg, about 0.75 to about 30 mg/kg, about1 to about 30 mg/kg, about 1.5 to about 30 mg/kg, about 2 to about 30mg/kg, about 2.5 to about 30 mg/kg, about 3 to about 30 mg/kg, about 3.5to about 30 mg/kg, about 4 to about 30 mg/kg, about 4.5 to about 30mg/kg, about 5 to about 30 mg/kg, about 7.5 to about 30 mg/kg, about 10to about 30 mg/kg, about 15 to about 30 mg/kg, about 20 to about 30mg/kg, about 20 to about 30 mg/kg, about 25 to about 30 mg/kg, about 0.5to about 20 mg/kg, about 0.75 to about 20 mg/kg, about 1 to about 20mg/kg, about 1.5 to about 20 mg/kg, about 2 to about 20 mg/kg, about 2.5to about 20 mg/kg, about 3 to about 20 mg/kg, about 3.5 to about 20mg/kg, about 4 to about 20 mg/kg, about 4.5 to about 20 mg/kg, about 5to about 20 mg/kg, about 7.5 to about 20 mg/kg, about 10 to about 20mg/kg, or about 15 to about 20 mg/kg. In one embodiment, the dsRNA isadministered at a dose of about 10 mg/kg to about 30 mg/kg. Values andranges intermediate to the recited values are also intended to be partof this invention.

For example, subjects can be administered, e.g., subcutaneously,intramuscularly, or intravenously, a single therapeutic amount of iRNA,such as about 0.1, 0.125, 0.15, 0.175, 0.2, 0.225, 0.25, 0.275, 0.3,0.325, 0.35, 0.375, 0.4, 0.425, 0.45, 0.475, 0.5, 0.525, 0.55, 0.575,0.6, 0.625, 0.65, 0.675, 0.7, 0.725, 0.75, 0.775, 0.8, 0.825, 0.85,0.875, 0.9, 0.925, 0.95, 0.975, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7,1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2,3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7,4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2,6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7,7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2,9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10, 10.5, 11, 11.5, 12, 12.5, 13,13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, 20,20.5, 21, 21.5, 22, 22.5, 23, 23.5, 24, 24.5, 25, 25.5, 26, 26.5, 27,27.5, 28, 28.5, 29, 29.5, 30, 31, 32, 33, 34, 34, 35, 36, 37, 38, 39,40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or about 50 mg/kg. Values andranges intermediate to the recited values are also intended to be partof this invention.

In some embodiments, subjects are administered, e.g., subcutaneously,intramuscularly, or intravenously, multiple doses of a therapeuticamount of iRNA, such as a dose about 0.1, 0.125, 0.15, 0.175, 0.2,0.225, 0.25, 0.275, 0.3, 0.325, 0.35, 0.375, 0.4, 0.425, 0.45, 0.475,0.5, 0.525, 0.55, 0.575, 0.6, 0.625, 0.65, 0.675, 0.7, 0.725, 0.75,0.775, 0.8, 0.825, 0.85, 0.875, 0.9, 0.925, 0.95, 0.975, 1, 1.1, 1.2,1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7,2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2,4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7,5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2,7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7,8.8, 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10, 10.5, 11,11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18,18.5, 19, 19.5, 20, 20.5, 21, 21.5, 22, 22.5, 23, 23.5, 24, 24.5, 25,25.5, 26, 26.5, 27, 27.5, 28, 28.5, 29, 29.5, 30, 31, 32, 33, 34, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or about 50mg/kg. A multi-dose regimine may include administration of a therapeuticamount of iRNA daily, such as for two days, three days, four days, fivedays, six days, seven days, or longer.

In other embodiments, subjects are administered, e.g., subcutaneously,intramuscularly, or intravenously, a repeat dose of a therapeutic amountof iRNA, such as a dose about 0.1, 0.125, 0.15, 0.175, 0.2, 0.225, 0.25,0.275, 0.3, 0.325, 0.35, 0.375, 0.4, 0.425, 0.45, 0.475, 0.5, 0.525,0.55, 0.575, 0.6, 0.625, 0.65, 0.675, 0.7, 0.725, 0.75, 0.775, 0.8,0.825, 0.85, 0.875, 0.9, 0.925, 0.95, 0.975, 1, 1.1, 1.2, 1.3, 1.4, 1.5,1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3,3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5,4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6,6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5,7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9,9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10, 10.5, 11, 11.5, 12,12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19,19.5, 20, 20.5, 21, 21.5, 22, 22.5, 23, 23.5, 24, 24.5, 25, 25.5, 26,26.5, 27, 27.5, 28, 28.5, 29, 29.5, 30, 31, 32, 33, 34, 34, 35, 36, 37,38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or about 50 mg/kg. Arepeat-dose regimine may include administration of a therapeutic amountof iRNA on a regular basis, such as every other day, every third day,every fourth day, twice a week, once a week, every other week, or once amonth.

In certain embodiments, for example, when a composition of the inventioncomprises a dsRNA as described herein and a lipid, subjects can beadministered a therapeutic amount of iRNA, such as about 0.01 mg/kg toabout 5 mg/kg, about 0.01 mg/kg to about 10 mg/kg, about 0.05 mg/kg toabout 5 mg/kg, about 0.05 mg/kg to about 10 mg/kg, about 0.1 mg/kg toabout 5 mg/kg, about 0.1 mg/kg to about 10 mg/kg, about 0.2 mg/kg toabout 5 mg/kg, about 0.2 mg/kg to about 10 mg/kg, about 0.3 mg/kg toabout 5 mg/kg, about 0.3 mg/kg to about 10 mg/kg, about 0.4 mg/kg toabout 5 mg/kg, about 0.4 mg/kg to about 10 mg/kg, about 0.5 mg/kg toabout 5 mg/kg, about 0.5 mg/kg to about 10 mg/kg, about 1 mg/kg to about5 mg/kg, about 1 mg/kg to about 10 mg/kg, about 1.5 mg/kg to about 5mg/kg, about 1.5 mg/kg to about 10 mg/kg, about 2 mg/kg to about about2.5 mg/kg, about 2 mg/kg to about 10 mg/kg, about 3 mg/kg to about 5mg/kg, about 3 mg/kg to about 10 mg/kg, about 3.5 mg/kg to about 5mg/kg, about 4 mg/kg to about 5 mg/kg, about 4.5 mg/kg to about 5 mg/kg,about 4 mg/kg to about 10 mg/kg, about 4.5 mg/kg to about 10 mg/kg,about 5 mg/kg to about 10 mg/kg, about 5.5 mg/kg to about 10 mg/kg,about 6 mg/kg to about 10 mg/kg, about 6.5 mg/kg to about 10 mg/kg,about 7 mg/kg to about 10 mg/kg, about 7.5 mg/kg to about 10 mg/kg,about 8 mg/kg to about 10 mg/kg, about 8.5 mg/kg to about 10 mg/kg,about 9 mg/kg to about 10 mg/kg, or about 9.5 mg/kg to about 10 mg/kg.Values and ranges intermediate to the recited values are also intendedto be part of this invention.

For example, the dsRNA may be administered at a dose of about 0.1, 0.2,0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7,1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2,3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7,4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2,6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7,7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2,9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or about 10 mg/kg. Values and rangesintermediate to the recited values are also intended to be part of thisinvention.

In certain embodiments of the invention, for example, when adouble-stranded RNAi agent includes a modification (e.g., one or moremotifs of three identical modifications on three consecutivenucleotides), including one such motif at or near the cleavage site ofthe agent, six phosphorothioate linkages, and a ligand, such an agent isadministered at a dose of about 0.01 to about 0.5 mg/kg, about 0.01 toabout 0.4 mg/kg, about 0.01 to about 0.3 mg/kg, about 0.01 to about 0.2mg/kg, about 0.01 to about 0.1 mg/kg, about 0.01 mg/kg to about 0.09mg/kg, about 0.01 mg/kg to about 0.08 mg/kg, about 0.01 mg/kg to about0.07 mg/kg, about 0.01 mg/kg to about 0.06 mg/kg, about 0.01 mg/kg toabout 0.05 mg/kg, about 0.02 to about 0.5 mg/kg, about 0.02 to about 0.4mg/kg, about 0.02 to about 0.3 mg/kg, about 0.02 to about 0.2 mg/kg,about 0.02 to about 0.1 mg/kg, about 0.02 mg/kg to about 0.09 mg/kg,about 0.02 mg/kg to about 0.08 mg/kg, about 0.02 mg/kg to about 0.07mg/kg, about 0.02 mg/kg to about 0.06 mg/kg, about 0.02 mg/kg to about0.05 mg/kg, about 0.03 to about 0.5 mg/kg, about 0.03 to about 0.4mg/kg, about 0.03 to about 0.3 mg/kg, about 0.03 to about 0.2 mg/kg,about 0.03 to about 0.1 mg/kg, about 0.03 mg/kg to about 0.09 mg/kg,about 0.03 mg/kg to about 0.08 mg/kg, about 0.03 mg/kg to about 0.07mg/kg, about 0.03 mg/kg to about 0.06 mg/kg, about 0.03 mg/kg to about0.05 mg/kg, about 0.04 to about 0.5 mg/kg, about 0.04 to about 0.4mg/kg, about 0.04 to about 0.3 mg/kg, about 0.04 to about 0.2 mg/kg,about 0.04 to about 0.1 mg/kg, about 0.04 mg/kg to about 0.09 mg/kg,about 0.04 mg/kg to about 0.08 mg/kg, about 0.04 mg/kg to about 0.07mg/kg, about 0.04 mg/kg to about 0.06 mg/kg, about 0.05 to about 0.5mg/kg, about 0.05 to about 0.4 mg/kg, about 0.05 to about 0.3 mg/kg,about 0.05 to about 0.2 mg/kg, about 0.05 to about 0.1 mg/kg, about 0.05mg/kg to about 0.09 mg/kg, about 0.05 mg/kg to about 0.08 mg/kg, orabout 0.05 mg/kg to about 0.07 mg/kg. Values and ranges intermediate tothe foregoing recited values are also intended to be part of thisinvention, e.g., the RNAi agent may be administered to the subject at adose of about 0.015 mg/kg to about 0.45 mg/kg.

For example, the RNAi agent, e.g., RNAi agent in a pharmaceuticalcomposition, may be administered at a dose of about 0.01 mg/kg, 0.0125mg/kg, 0.015 mg/kg, 0.0175 mg/kg, 0.02 mg/kg, 0.0225 mg/kg, 0.025 mg/kg,0.0275 mg/kg, 0.03 mg/kg, 0.0325 mg/kg, 0.035 mg/kg, 0.0375 mg/kg, 0.04mg/kg, 0.0425 mg/kg, 0.045 mg/kg, 0.0475 mg/kg, 0.05 mg/kg, 0.0525mg/kg, 0.055 mg/kg, 0.0575 mg/kg, 0.06 mg/kg, 0.0625 mg/kg, 0.065 mg/kg,0.0675 mg/kg, 0.07 mg/kg, 0.0725 mg/kg, 0.075 mg/kg, 0.0775 mg/kg, 0.08mg/kg, 0.0825 mg/kg, 0.085 mg/kg, 0.0875 mg/kg, 0.09 mg/kg, 0.0925mg/kg, 0.095 mg/kg, 0.0975 mg/kg, 0.1 mg/kg, 0.125 mg/kg, 0.15 mg/kg,0.175 mg/kg, 0.2 mg/kg, 0.225 mg/kg, 0.25 mg/kg, 0.275 mg/kg, 0.3 mg/kg,0.325 mg/kg, 0.35 mg/kg, 0.375 mg/kg, 0.4 mg/kg, 0.425 mg/kg, 0.45mg/kg, 0.475 mg/kg, or about 0.5 mg/kg. Values intermediate to theforegoing recited values are also intended to be part of this invention.

In some embodiments, the RNAi agent is administered as a fixed dose ofbetween about 100 mg to about 900 mg, e.g., between about 100 mg toabout 850 mg, between about 100 mg to about 800 mg, between about 100 mgto about 750 mg, between about 100 mg to about 700 mg, between about 100mg to about 650 mg, between about 100 mg to about 600 mg, between about100 mg to about 550 mg, between about 100 mg to about 500 mg, betweenabout 200 mg to about 850 mg, between about 200 mg to about 800 mg,between about 200 mg to about 750 mg, between about 200 mg to about 700mg, between about 200 mg to about 650 mg, between about 200 mg to about600 mg, between about 200 mg to about 550 mg, between about 200 mg toabout 500 mg, between about 300 mg to about 850 mg, between about 300 mgto about 800 mg, between about 300 mg to about 750 mg, between about 300mg to about 700 mg, between about 300 mg to about 650 mg, between about300 mg to about 600 mg, between about 300 mg to about 550 mg, betweenabout 300 mg to about 500 mg, between about 400 mg to about 850 mg,between about 400 mg to about 800 mg, between about 400 mg to about 750mg, between about 400 mg to about 700 mg, between about 400 mg to about650 mg, between about 400 mg to about 600 mg, between about 400 mg toabout 550 mg, or between about 400 mg to about 500 mg.

In some embodiments, the RNAi agent is administered as a fixed dose ofabout 100 mg, about 125 mg, about 150 mg, about 175 mg, 200 mg, about225 mg, about 250 mg, about 275 mg, about 300 mg, about 325 mg, about350 mg, about 375 mg, about 400 mg, about 425 mg, about 450 mg, about475 mg, about 500 mg, about 525 mg, about 550 mg, about 575 mg, about600 mg, about 625 mg, about 650 mg, about 675 mg, about 700 mg, about725 mg, about 750 mg, about 775 mg, about 800 mg, about 825 mg, about850 mg, about 875 mg, or about 900 mg.

The pharmaceutical composition can be administered by intravenousinfusion over a period of time, such as over a 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, and 21, 22, 23, 24, or about a 25minute period. The administration may be repeated, for example, on aregular basis, such as weekly, biweekly (i.e., every two weeks) for onemonth, two months, three months, four months or longer. After an initialtreatment regimen, the treatments can be administered on a less frequentbasis. For example, after administration weekly or biweekly for threemonths, administration can be repeated once per month, for six months ora year or longer.

The pharmaceutical composition can be administered once daily, or theiRNA can be administered as two, three, or more sub-doses at appropriateintervals throughout the day or even using continuous infusion ordelivery through a controlled release formulation. In that case, theiRNA contained in each sub-dose must be correspondingly smaller in orderto achieve the total daily dosage. The dosage unit can also becompounded for delivery over several days, e.g., using a conventionalsustained release formulation which provides sustained release of theiRNA over a several day period. Sustained release formulations are wellknown in the art and are particularly useful for delivery of agents at aparticular site, such as could be used with the agents of the presentinvention. In this embodiment, the dosage unit contains a correspondingmultiple of the daily dose.

In other embodiments, a single dose of the pharmaceutical compositionscan be long lasting, such that subsequent doses are administered at notmore than 3, 4, or 5 day intervals, or at not more than 1, 2, 3, or 4week intervals. In some embodiments of the invention, a single dose ofthe pharmaceutical compositions of the invention is administered onceper week. In other embodiments of the invention, a single dose of thepharmaceutical compositions of the invention is administered bi-monthly.In some embodiments of the invention, a single dose of thepharmaceutical compositions of the invention is administered once permonth, once every other month, or once quarterly (i.e., every threemonths).

The skilled artisan will appreciate that certain factors can influencethe dosage and timing required to effectively treat a subject, includingbut not limited to the severity of the disease or disorder, previoustreatments, the general health and/or age of the subject, and otherdiseases present. Moreover, treatment of a subject with atherapeutically effective amount of a composition can include a singletreatment or a series of treatments. Estimates of effective dosages andin vivo half-lives for the individual iRNAs encompassed by the inventioncan be made using conventional methodologies or on the basis of in vivotesting using an appropriate animal model, as described elsewhereherein.

The pharmaceutical compositions of the present invention can beadministered in a number of ways depending upon whether local orsystemic treatment is desired and upon the area to be treated.Administration can be topical (e.g., by a transdermal patch), pulmonary,e.g., by inhalation or insufflation of powders or aerosols, including bynebulizer; intratracheal, intranasal, epidermal and transdermal, oral orparenteral. Parenteral administration includes intravenous,intraarterial, subcutaneous, intraperitoneal or intramuscular injectionor infusion; subdermal, e.g., via an implanted device; or intracranial,e.g., by intraparenchymal, intrathecal or intraventricular,administration.

The iRNA can be delivered in a manner to target a particular tissue,such as the liver (e.g., the hepatocytes of the liver).

Pharmaceutical compositions and formulations for topical administrationcan include transdermal patches, ointments, lotions, creams, gels,drops, suppositories, sprays, liquids and powders. Conventionalpharmaceutical carriers, aqueous, powder or oily bases, thickeners andthe like can be necessary or desirable. Coated condoms, gloves and thelike can also be useful. Suitable topical formulations include those inwhich the iRNAs featured in the invention are in admixture with atopical delivery agent such as lipids, liposomes, fatty acids, fattyacid esters, steroids, chelating agents and surfactants. Suitable lipidsand liposomes include neutral (e.g., dioleoylphosphatidyl DOPEethanolamine, dimyristoylphosphatidyl choline DMPC,distearolyphosphatidyl choline) negative (e.g., dimyristoylphosphatidylglycerol DMPG) and cationic (e.g., dioleoyltetramethylaminopropyl DOTAPand dioleoylphosphatidyl ethanolamine DOTMA). iRNAs featured in theinvention can be encapsulated within liposomes or can form complexesthereto, in particular to cationic liposomes. Alternatively, iRNAs canbe complexed to lipids, in particular to cationic lipids. Suitable fattyacids and esters include but are not limited to arachidonic acid, oleicacid, eicosanoic acid, lauric acid, caprylic acid, capric acid, myristicacid, palmitic acid, stearic acid, linoleic acid, linolenic acid,dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1-monocaprate,1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or aC₁₋₂₀ alkyl ester (e.g., isopropylmyristate IPM), monoglyceride,diglyceride or pharmaceutically acceptable salt thereof). Topicalformulations are described in detail in U.S. Pat. No. 6,747,014, whichis incorporated herein by reference.

A. iRNA Formulations Comprising Membranous Molecular Assemblies

An iRNA for use in the compositions and methods of the invention can beformulated for delivery in a membranous molecular assembly, e.g., aliposome or a micelle. As used herein, the term “liposome” refers to avesicle composed of amphiphilic lipids arranged in at least one bilayer,e.g., one bilayer or a plurality of bilayers. Liposomes includeunilamellar and multilamellar vesicles that have a membrane formed froma lipophilic material and an aqueous interior. The aqueous portioncontains the iRNA composition. The lipophilic material isolates theaqueous interior from an aqueous exterior, which typically does notinclude the iRNA composition, although in some examples, it may.Liposomes are useful for the transfer and delivery of active ingredientsto the site of action. Because the liposomal membrane is structurallysimilar to biological membranes, when liposomes are applied to a tissue,the liposomal bilayer fuses with bilayer of the cellular membranes. Asthe merging of the liposome and cell progresses, the internal aqueouscontents that include the iRNA are delivered into the cell where theiRNA can specifically bind to a target RNA and can mediate iRNA. In somecases the liposomes are also specifically targeted, e.g., to direct theiRNA to particular cell types.

A liposome containing an iRNA agent can be prepared by a variety ofmethods. In one example, the lipid component of a liposome is dissolvedin a detergent so that micelles are formed with the lipid component. Forexample, the lipid component can be an amphipathic cationic lipid orlipid conjugate. The detergent can have a high critical micelleconcentration and may be nonionic. Exemplary detergents include cholate,CHAPS, octylglucoside, deoxycholate, and lauroyl sarcosine. The iRNAagent preparation is then added to the micelles that include the lipidcomponent. The cationic groups on the lipid interact with the iRNA agentand condense around the iRNA agent to form a liposome. Aftercondensation, the detergent is removed, e.g., by dialysis, to yield aliposomal preparation of iRNA agent.

If necessary a carrier compound that assists in condensation can beadded during the condensation reaction, e.g., by controlled addition.For example, the carrier compound can be a polymer other than a nucleicacid (e.g., spermine or spermidine). pH can also adjusted to favorcondensation.

Methods for producing stable polynucleotide delivery vehicles, whichincorporate a polynucleotide/cationic lipid complex as structuralcomponents of the delivery vehicle, are further described in, e.g., WO96/37194, the entire contents of which are incorporated herein byreference. Liposome formation can also include one or more aspects ofexemplary methods described in Felgner, P. L. et al., Proc. Natl. Acad.Sci., USA 8:7413-7417, 1987; U.S. Pat. Nos. 4,897,355; 5,171,678;Bangham, et al. M. Mol. Biol. 23:238, 1965; Olson, et al. Biochim.Biophys. Acta 557:9, 1979; Szoka, et al. Proc. Natl. Acad. Sci. 75:4194, 1978; Mayhew, et al. Biochim. Biophys. Acta 775:169, 1984; Kim, etal. Biochim. Biophys. Acta 728:339, 1983; and Fukunaga, et al.Endocrinol. 115:757, 1984. Commonly used techniques for preparing lipidaggregates of appropriate size for use as delivery vehicles includesonication and freeze-thaw plus extrusion (see, e.g., Mayer, et al.Biochim. Biophys. Acta 858:161, 1986). Microfluidization can be usedwhen consistently small (50 to 200 nm) and relatively uniform aggregatesare desired (Mayhew, et al. Biochim. Biophys. Acta 775:169, 1984). Thesemethods are readily adapted to packaging iRNA agent preparations intoliposomes.

Liposomes fall into two broad classes. Cationic liposomes are positivelycharged liposomes which interact with the negatively charged nucleicacid molecules to form a stable complex. The positively charged nucleicacid/liposome complex binds to the negatively charged cell surface andis internalized in an endosome. Due to the acidic pH within theendosome, the liposomes are ruptured, releasing their contents into thecell cytoplasm (Wang et al., Biochem. Biophys. Res. Commun., 1987, 147,980-985).

Liposomes which are pH-sensitive or negatively-charged, entrap nucleicacids rather than complex with it. Since both the nucleic acid and thelipid are similarly charged, repulsion rather than complex formationoccurs. Nevertheless, some nucleic acid is entrapped within the aqueousinterior of these liposomes. pH-sensitive liposomes have been used todeliver nucleic acids encoding the thymidine kinase gene to cellmonolayers in culture. Expression of the exogenous gene was detected inthe target cells (Zhou et al., Journal of Controlled Release, 1992, 19,269-274).

One major type of liposomal composition includes phospholipids otherthan naturally-derived phosphatidylcholine. Neutral liposomecompositions, for example, can be formed from dimyristoylphosphatidylcholine (DMPC) or dipalmitoyl phosphatidylcholine (DPPC).Anionic liposome compositions generally are formed from dimyristoylphosphatidylglycerol, while anionic fusogenic liposomes are formedprimarily from dioleoyl phosphatidylethanolamine (DOPE). Another type ofliposomal composition is formed from phosphatidylcholine (PC) such as,for example, soybean PC, and egg PC. Another type is formed frommixtures of phospholipid and/or phosphatidylcholine and/or cholesterol.

Examples of other methods to introduce liposomes into cells in vitro andin vivo include U.S. Pat. Nos. 5,283,185; 5,171,678; WO 94/00569; WO93/24640; WO 91/16024; Felgner, J. Biol. Chem. 269:2550, 1994; Nabel,Proc. Natl. Acad. Sci. 90:11307, 1993; Nabel, Human Gene Ther. 3:649,1992; Gershon, Biochem. 32:7143, 1993; and Strauss EMBO J. 11:417, 1992.

Non-ionic liposomal systems have also been examined to determine theirutility in the delivery of drugs to the skin, in particular systemscomprising non-ionic surfactant and cholesterol. Non-ionic liposomalformulations comprising Novasome™ I (glyceryldilaurate/cholesterol/polyoxyethylene-10-stearyl ether) and Novasome™ II(glyceryl distearate/cholesterol/polyoxyethylene-10-stearyl ether) wereused to deliver cyclosporin-A into the dermis of mouse skin. Resultsindicated that such non-ionic liposomal systems were effective infacilitating the deposition of cyclosporine A into different layers ofthe skin (Hu et al. S.T.P. Pharma. Sci., 1994, 4(6) 466).

Liposomes also include “sterically stabilized” liposomes, a term which,as used herein, refers to liposomes comprising one or more specializedlipids that, when incorporated into liposomes, result in enhancedcirculation lifetimes relative to liposomes lacking such specializedlipids. Examples of sterically stabilized liposomes are those in whichpart of the vesicle-forming lipid portion of the liposome (A) comprisesone or more glycolipids, such as monosialoganglioside G_(M1), or (B) isderivatized with one or more hydrophilic polymers, such as apolyethylene glycol (PEG) moiety. While not wishing to be bound by anyparticular theory, it is thought in the art that, at least forsterically stabilized liposomes containing gangliosides, sphingomyelin,or PEG-derivatized lipids, the enhanced circulation half-life of thesesterically stabilized liposomes derives from a reduced uptake into cellsof the reticuloendothelial system (RES) (Allen et al., FEBS Letters,1987, 223, 42; Wu et al., Cancer Research, 1993, 53, 3765).

Various liposomes comprising one or more glycolipids are known in theart. Papahadjopoulos et al. (Ann. N.Y. Acad. Sci., 1987, 507, 64)reported the ability of monosialoganglioside G_(M1), galactocerebrosidesulfate and phosphatidylinositol to improve blood half-lives ofliposomes. These findings were expounded upon by Gabizon et al. (Proc.Natl. Acad. Sci. U.S.A., 1988, 85, 6949). U.S. Pat. No. 4,837,028 and WO88/04924, both to Allen et al., disclose liposomes comprising (1)sphingomyelin and (2) the ganglioside G_(M1) or a galactocerebrosidesulfate ester. U.S. Pat. No. 5,543,152 (Webb et al.) discloses liposomescomprising sphingomyelin. Liposomes comprising1,2-sn-dimyristoylphosphatidylcholine are disclosed in WO 97/13499 (Limet al).

In one embodiment, cationic liposomes are used. Cationic liposomespossess the advantage of being able to fuse to the cell membrane.Non-cationic liposomes, although not able to fuse as efficiently withthe plasma membrane, are taken up by macrophages in vivo and can be usedto deliver iRNA agents to macrophages.

Further advantages of liposomes include: liposomes obtained from naturalphospholipids are biocompatible and biodegradable; liposomes canincorporate a wide range of water and lipid soluble drugs; liposomes canprotect encapsulated iRNA agents in their internal compartments frommetabolism and degradation (Rosoff, in “Pharmaceutical Dosage Forms,”Lieberman, Rieger and Banker (Eds.), 1988, volume 1, p. 245). Importantconsiderations in the preparation of liposome formulations are the lipidsurface charge, vesicle size and the aqueous volume of the liposomes.

A positively charged synthetic cationic lipid,N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA)can be used to form small liposomes that interact spontaneously withnucleic acid to form lipid-nucleic acid complexes which are capable offusing with the negatively charged lipids of the cell membranes oftissue culture cells, resulting in delivery of iRNA agent (see, e.g.,Felgner, P. L. et al., Proc. Natl. Acad. Sci., USA 8:7413-7417, 1987 andU.S. Pat. No. 4,897,355 for a description of DOTMA and its use withDNA).

A DOTMA analogue, 1,2-bis(oleoyloxy)-3-(trimethylammonia)propane (DOTAP)can be used in combination with a phospholipid to form DNA-complexingvesicles. Lipofectin™ Bethesda Research Laboratories, Gaithersburg, Md.)is an effective agent for the delivery of highly anionic nucleic acidsinto living tissue culture cells that comprise positively charged DOTMAliposomes which interact spontaneously with negatively chargedpolynucleotides to form complexes. When enough positively chargedliposomes are used, the net charge on the resulting complexes is alsopositive. Positively charged complexes prepared in this wayspontaneously attach to negatively charged cell surfaces, fuse with theplasma membrane, and efficiently deliver functional nucleic acids into,for example, tissue culture cells. Another commercially availablecationic lipid, 1,2-bis(oleoyloxy)-3,3-(trimethylammonia)propane(“DOTAP”) (Boehringer Mannheim, Indianapolis, Ind.) differs from DOTMAin that the oleoyl moieties are linked by ester, rather than etherlinkages.

Other reported cationic lipid compounds include those that have beenconjugated to a variety of moieties including, for example,carboxyspermine which has been conjugated to one of two types of lipidsand includes compounds such as 5-carboxyspermylglycine dioctaoleoylamide(“DOGS”) (Transfectam™, Promega, Madison, Wis.) anddipalmitoylphosphatidylethanolamine 5-carboxyspermyl-amide (“DPPES”)(see, e.g., U.S. Pat. No. 5,171,678).

Another cationic lipid conjugate includes derivatization of the lipidwith cholesterol (“DC-Chol”) which has been formulated into liposomes incombination with DOPE (See, Gao, X. and Huang, L., Biochim. Biophys.Res. Commun. 179:280, 1991). Lipopolylysine, made by conjugatingpolylysine to DOPE, has been reported to be effective for transfectionin the presence of serum (Zhou, X. et al., Biochim. Biophys. Acta1065:8, 1991). For certain cell lines, these liposomes containingconjugated cationic lipids, are said to exhibit lower toxicity andprovide more efficient transfection than the DOTMA-containingcompositions. Other commercially available cationic lipid productsinclude DMRIE and DMRIE-HP (Vical, La Jolla, Calif.) and Lipofectamine(DOSPA) (Life Technology, Inc., Gaithersburg, Md.). Other cationiclipids suitable for the delivery of oligonucleotides are described in WO98/39359 and WO 96/37194.

Liposomal formulations are particularly suited for topicaladministration, liposomes present several advantages over otherformulations. Such advantages include reduced side effects related tohigh systemic absorption of the administered drug, increasedaccumulation of the administered drug at the desired target, and theability to administer iRNA agent into the skin. In some implementations,liposomes are used for delivering iRNA agent to epidermal cells and alsoto enhance the penetration of iRNA agent into dermal tissues, e.g., intoskin. For example, the liposomes can be applied topically. Topicaldelivery of drugs formulated as liposomes to the skin has beendocumented (see, e.g., Weiner et al., Journal of Drug Targeting, 1992,vol. 2, 405-410 and du Plessis et al., Antiviral Research, 18, 1992,259-265; Mannino, R. J. and Fould-Fogerite, S., Biotechniques 6:682-690,1988; Itani, T. et al. Gene 56:267-276. 1987; Nicolau, C. et al. Meth.Enz. 149:157-176, 1987; Straubinger, R. M. and Papahadjopoulos, D. Meth.Enz. 101:512-527, 1983; Wang, C. Y. and Huang, L., Proc. Natl. Acad.Sci. USA 84:7851-7855, 1987).

Non-ionic liposomal systems have also been examined to determine theirutility in the delivery of drugs to the skin, in particular systemscomprising non-ionic surfactant and cholesterol. Non-ionic liposomalformulations comprising Novasome I (glyceryldilaurate/cholesterol/polyoxyethylene-10-stearyl ether) and Novasome II(glyceryl distearate/cholesterol/polyoxyethylene-10-stearyl ether) wereused to deliver a drug into the dermis of mouse skin. Such formulationswith iRNA agent are useful for treating a dermatological disorder.

Liposomes that include iRNA can be made highly deformable. Suchdeformability can enable the liposomes to penetrate through pore thatare smaller than the average radius of the liposome. For example,transfersomes are a type of deformable liposomes.

Transferosomes can be made by adding surface edge activators, usuallysurfactants, to a standard liposomal composition. Transfersomes thatinclude iRNA agent can be delivered, for example, subcutaneously byinfection in order to deliver iRNA agent to keratinocytes in the skin.In order to cross intact mammalian skin, lipid vesicles must passthrough a series of fine pores, each with a diameter less than 50 nm,under the influence of a suitable transdermal gradient. In addition, dueto the lipid properties, these transferosomes can be self-optimizing(adaptive to the shape of pores, e.g., in the skin), self-repairing, andcan frequently reach their targets without fragmenting, and oftenself-loading.

Other formulations amenable to the present invention are described in,for example, PCT Publication No. WO 2008/042973, the entire contents ofwhich are incorporated herein by reference.

Transfersomes are yet another type of liposomes, and are highlydeformable lipid aggregates which are attractive candidates for drugdelivery vehicles. Transfersomes can be described as lipid dropletswhich are so highly deformable that they are easily able to penetratethrough pores which are smaller than the droplet. Transfersomes areadaptable to the environment in which they are used, e.g., they areself-optimizing (adaptive to the shape of pores in the skin),self-repairing, frequently reach their targets without fragmenting, andoften self-loading. To make transfersomes it is possible to add surfaceedge-activators, usually surfactants, to a standard liposomalcomposition. Transfersomes have been used to deliver serum albumin tothe skin. The transfersome-mediated delivery of serum albumin has beenshown to be as effective as subcutaneous injection of a solutioncontaining serum albumin.

Surfactants find wide application in formulations such as emulsions(including microemulsions) and liposomes. The most common way ofclassifying and ranking the properties of the many different types ofsurfactants, both natural and synthetic, is by the use of thehydrophile/lipophile balance (HLB). The nature of the hydrophilic group(also known as the “head”) provides the most useful means forcategorizing the different surfactants used in formulations (Rieger, in“Pharmaceutical Dosage Forms”, Marcel Dekker, Inc., New York, N.Y.,1988, p. 285).

If the surfactant molecule is not ionized, it is classified as anonionic surfactant. Nonionic surfactants find wide application inpharmaceutical and cosmetic products and are usable over a wide range ofpH values. In general their HLB values range from 2 to about 18depending on their structure. Nonionic surfactants include nonionicesters such as ethylene glycol esters, propylene glycol esters, glycerylesters, polyglyceryl esters, sorbitan esters, sucrose esters, andethoxylated esters. Nonionic alkanolamides and ethers such as fattyalcohol ethoxylates, propoxylated alcohols, and ethoxylated/propoxylatedblock polymers are also included in this class. The polyoxyethylenesurfactants are the most popular members of the nonionic surfactantclass.

If the surfactant molecule carries a negative charge when it isdissolved or dispersed in water, the surfactant is classified asanionic. Anionic surfactants include carboxylates such as soaps, acyllactylates, acyl amides of amino acids, esters of sulfuric acid such asalkyl sulfates and ethoxylated alkyl sulfates, sulfonates such as alkylbenzene sulfonates, acyl isethionates, acyl taurates andsulfosuccinates, and phosphates. The most important members of theanionic surfactant class are the alkyl sulfates and the soaps.

If the surfactant molecule carries a positive charge when it isdissolved or dispersed in water, the surfactant is classified ascationic. Cationic surfactants include quaternary ammonium salts andethoxylated amines. The quaternary ammonium salts are the most usedmembers of this class.

If the surfactant molecule has the ability to carry either a positive ornegative charge, the surfactant is classified as amphoteric. Amphotericsurfactants include acrylic acid derivatives, substituted alkylamides,N-alkylbetaines and phosphatides.

The use of surfactants in drug products, formulations and in emulsionshas been reviewed (Rieger, in “Pharmaceutical Dosage Forms”, MarcelDekker, Inc., New York, N.Y., 1988, p. 285).

The iRNA for use in the methods of the invention can also be provided asmicellar formulations. “Micelles” are defined herein as a particulartype of molecular assembly in which amphipathic molecules are arrangedin a spherical structure such that all the hydrophobic portions of themolecules are directed inward, leaving the hydrophilic portions incontact with the surrounding aqueous phase. The converse arrangementexists if the environment is hydrophobic.

A mixed micellar formulation suitable for delivery through transdermalmembranes may be prepared by mixing an aqueous solution of the siRNAcomposition, an alkali metal C₈ to C₂₂ alkyl sulphate, and a micelleforming compounds. Exemplary micelle forming compounds include lecithin,hyaluronic acid, pharmaceutically acceptable salts of hyaluronic acid,glycolic acid, lactic acid, chamomile extract, cucumber extract, oleicacid, linoleic acid, linolenic acid, monoolein, monooleates,monolaurates, borage oil, evening of primrose oil, menthol, trihydroxyoxo cholanyl glycine and pharmaceutically acceptable salts thereof,glycerin, polyglycerin, lysine, polylysine, triolein, polyoxyethyleneethers and analogues thereof, polidocanol alkyl ethers and analoguesthereof, chenodeoxycholate, deoxycholate, and mixtures thereof. Themicelle forming compounds may be added at the same time or afteraddition of the alkali metal alkyl sulphate. Mixed micelles will formwith substantially any kind of mixing of the ingredients but vigorousmixing in order to provide smaller size micelles.

In one method a first micellar composition is prepared which containsthe siRNA composition and at least the alkali metal alkyl sulphate. Thefirst micellar composition is then mixed with at least three micelleforming compounds to form a mixed micellar composition. In anothermethod, the micellar composition is prepared by mixing the siRNAcomposition, the alkali metal alkyl sulphate and at least one of themicelle forming compounds, followed by addition of the remaining micelleforming compounds, with vigorous mixing.

Phenol and/or m-cresol may be added to the mixed micellar composition tostabilize the formulation and protect against bacterial growth.Alternatively, phenol and/or m-cresol may be added with the micelleforming ingredients. An isotonic agent such as glycerin may also beadded after formation of the mixed micellar composition.

For delivery of the micellar formulation as a spray, the formulation canbe put into an aerosol dispenser and the dispenser is charged with apropellant. The propellant, which is under pressure, is in liquid formin the dispenser. The ratios of the ingredients are adjusted so that theaqueous and propellant phases become one, i.e., there is one phase. Ifthere are two phases, it is necessary to shake the dispenser prior todispensing a portion of the contents, e.g., through a metered valve. Thedispensed dose of pharmaceutical agent is propelled from the meteredvalve in a fine spray.

Propellants may include hydrogen-containing chlorofluorocarbons,hydrogen-containing fluorocarbons, dimethyl ether and diethyl ether. Incertain embodiments, HFA 134a (1,1,1,2 tetrafluoroethane) may be used.

The specific concentrations of the essential ingredients can bedetermined by relatively straightforward experimentation. For absorptionthrough the oral cavities, it is often desirable to increase, e.g., atleast double or triple, the dosage for through injection oradministration through the gastrointestinal tract.

B. Lipid Particles

iRNAs, e.g., dsRNAs of in the invention may be fully encapsulated in alipid formulation, e.g., a LNP, or other nucleic acid-lipid particle.

As used herein, the term “LNP” refers to a stable nucleic acid-lipidparticle. LNPs typically contain a cationic lipid, a non-cationic lipid,and a lipid that prevents aggregation of the particle (e.g., a PEG-lipidconjugate). LNPs are extremely useful for systemic applications, as theyexhibit extended circulation lifetimes following intravenous (i.v.)injection and accumulate at distal sites (e.g., sites physicallyseparated from the administration site). LNPs include “pSPLP,” whichinclude an encapsulated condensing agent-nucleic acid complex as setforth in PCT Publication No. WO 00/03683. The particles of the presentinvention typically have a mean diameter of about 50 nm to about 150 nm,more typically about 60 nm to about 130 nm, more typically about 70 nmto about 110 nm, most typically about 70 nm to about 90 nm, and aresubstantially nontoxic. In addition, the nucleic acids when present inthe nucleic acid-lipid particles of the present invention are resistantin aqueous solution to degradation with a nuclease. Nucleic acid-lipidparticles and their method of preparation are disclosed in, e.g., U.S.Pat. Nos. 5,976,567; 5,981,501; 6,534,484; 6,586,410; 6,815,432; U.S.Publication No. 2010/0324120 and PCT Publication No. WO 96/40964.

In one embodiment, the lipid to drug ratio (mass/mass ratio) (e.g.,lipid to dsRNA ratio) will be in the range of from about 1:1 to about50:1, from about 1:1 to about 25:1, from about 3:1 to about 15:1, fromabout 4:1 to about 10:1, from about 5:1 to about 9:1, or about 6:1 toabout 9:1. Ranges intermediate to the above recited ranges are alsocontemplated to be part of the invention.

The cationic lipid can be, for example, N,N-dioleyl-N,N-dimethylammoniumchloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB),N—(I-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP),N—(I-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA),N,N-dimethyl-2,3-dioleyloxy)propylamine (DODMA),1,2-DiLinoleyloxy-N,N-dimethylaminopropane (DLinDMA),1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA),1,2-Dilinoleylcarbamoyloxy-3-dimethylaminopropane (DLin-C-DAP),1,2-Dilinoleyoxy-3-(dimethylamino)acetoxypropane (DLin-DAC),1,2-Dilinoleyoxy-3-morpholinopropane (DLin-MA),1,2-Dilinoleoyl-3-dimethylaminopropane (DLinDAP),1,2-Dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA),1-Linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP),1,2-Dilinoleyloxy-3-trimethylaminopropane chloride salt (DLin-TMA.Cl),1,2-Dilinoleoyl-3-trimethylaminopropane chloride salt (DLin-TAP.Cl),1,2-Dilinoleyloxy-3-(N-methylpiperazino)propane (DLin-MPZ), or3-(N,N-Dilinoleylamino)-1,2-propanediol (DLinAP),3-(N,N-Dioleylamino)-1,2-propanedio (DOAP),1,2-Dilinoleyloxo-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA),1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLinDMA),2,2-Dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA) oranalogs thereof,(3aR,5s,6aS)—N,N-dimethyl-2,2-di((9Z,12Z)-octadeca-9,12-dienyl)tetrahydro-3aH-cyclopenta[d][1,3]dioxol-5-amine(ALN100), (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl4-(dimethylamino)butanoate (MC3),1,1′-(2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethylazanediyl)didodecan-2-ol(Tech GI), or a mixture thereof. The cationic lipid can comprise fromabout 20 mol % to about 50 mol % or about 40 mol % of the total lipidpresent in the particle.

In another embodiment, the compound2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane can be used toprepare lipid-siRNA nanoparticles. Synthesis of2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane is described in U.S.provisional patent application No. 61/107,998 filed on Oct. 23, 2008,which is herein incorporated by reference.

In one embodiment, the lipid-siRNA particle includes 40% 2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane: 10% DSPC: 40%Cholesterol: 10% PEG-C-DOMG (mole percent) with a particle size of63.0±20 nm and a 0.027 siRNA/Lipid Ratio.

The ionizable/non-cationic lipid can be an anionic lipid or a neutrallipid including, but not limited to, distearoylphosphatidylcholine(DSPC), dioleoylphosphatidylcholine (DOPC),dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol(DOPG), dipalmitoylphosphatidylglycerol (DPPG),dioleoyl-phosphatidylethanolamine (DOPE),palmitoyloleoylphosphatidylcholine (POPC),palmitoyloleoylphosphatidylethanolamine (POPE),dioleoyl-phosphatidylethanolamine4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE),distearoyl-phosphatidyl-ethanolamine (DSPE), 16-O-monomethyl PE,16-O-dimethyl PE, 18-1-trans PE,1-stearoyl-2-oleoyl-phosphatidyethanolamine (SOPE), cholesterol, or amixture thereof. The non-cationic lipid can be from about 5 mol % toabout 90 mol %, about 10 mol %, or about 58 mol % if cholesterol isincluded, of the total lipid present in the particle.

The conjugated lipid that inhibits aggregation of particles can be, forexample, a polyethyleneglycol (PEG)-lipid including, without limitation,a PEG-diacylglycerol (DAG), a PEG-dialkyloxypropyl (DAA), aPEG-phospholipid, a PEG-ceramide (Cer), or a mixture thereof. ThePEG-DAA conjugate can be, for example, a PEG-dilauryloxypropyl (Ci₂), aPEG-dimyristyloxypropyl (Ci₄), a PEG-dipalmityloxypropyl (Ci₆), or aPEG-distearyloxypropyl (C]₈). The conjugated lipid that preventsaggregation of particles can be from 0 mol % to about 20 mol % or about2 mol % of the total lipid present in the particle.

In some embodiments, the nucleic acid-lipid particle further includescholesterol at, e.g., about 10 mol % to about 60 mol % or about 48 mol %of the total lipid present in the particle.

In one embodiment, the lipidoid ND98.4HCl (MW 1487) (see U.S. patentapplication Ser. No. 12/056,230, filed Mar. 26, 2008, which isincorporated herein by reference), Cholesterol (Sigma-Aldrich), andPEG-Ceramide C16 (Avanti Polar Lipids) can be used to preparelipid-dsRNA nanoparticles (i.e., LNP01 particles). Stock solutions ofeach in ethanol can be prepared as follows: ND98, 133 mg/ml;Cholesterol, 25 mg/ml, PEG-Ceramide C16, 100 mg/ml. The ND98,Cholesterol, and PEG-Ceramide C16 stock solutions can then be combinedin a, e.g., 42:48:10 molar ratio. The combined lipid solution can bemixed with aqueous dsRNA (e.g., in sodium acetate pH 5) such that thefinal ethanol concentration is about 35-45% and the final sodium acetateconcentration is about 100-300 mM. Lipid-dsRNA nanoparticles typicallyform spontaneously upon mixing. Depending on the desired particle sizedistribution, the resultant nanoparticle mixture can be extruded througha polycarbonate membrane (e.g., 100 nm cut-off) using, for example, athermobarrel extruder, such as Lipex Extruder (Northern Lipids, Inc). Insome cases, the extrusion step can be omitted. Ethanol removal andsimultaneous buffer exchange can be accomplished by, for example,dialysis or tangential flow filtration. Buffer can be exchanged with,for example, phosphate buffered saline (PBS) at about pH 7, e.g., aboutpH 6.9, about pH 7.0, about pH 7.1, about pH 7.2, about pH 7.3, or aboutpH 7.4.

LNP01 formulations are described, e.g., in International ApplicationPublication No. WO 2008/042973, which is hereby incorporated byreference.

Additional exemplary lipid-dsRNA formulations are described in Table 1.

TABLE 1 cationic lipid/non-cationic lipid/cholesterol/PEG-lipidIonizable/Cationic Lipid conjugate Lipid:siRNA ratio SNALP-11,2-Dilinolenyloxy-N,N- DLinDMA/DPPC/Cholesterol/ dimethylaminopropanePEG-cDMA (DLinDMA) (57.1/7.1/34.4/1.4) lipid:siRNA~7:1 2-XTC2,2-Dilinoleyl-4- XTC/DPPC/Cholesterol/ dimethylaminoethyl- PEG-cDMA[1,3]-dioxolane (XTC) 57.1/7.1/34.4/1.4 lipid:siRNA~7:1 LNP052,2-Dilinoleyl-4- XTC/DSPC/Cholesterol/ dimethylaminoethyl- PEG-DMG[1,3]-dioxolane (XTC) 57.5/7.5/31.5/3.5 lipid:siRNA~6:1 LNP062,2-Dilinoleyl-4- XTC/DSPC/Cholesterol/ dimethylaminoethyl- PEG-DMG[1,3]-dioxolane (XTC) 57.5/7.5/31.5/3.5 lipid:siRNA~11:1 LNP072,2-Dilinoleyl-4- XTC/DSPC/Cholesterol/ dimethylaminoethyl- PEG-DMG[1,3]-dioxolane (XTC) 60/7.5/31/1.5, lipid:siRNA~6:1 LNP082,2-Dilinoleyl-4- XTC/DSPC/Cholesterol/ dimethylaminoethyl- PEG-DMG[1,3]-dioxolane (XTC) 60/7.5/31/1.5, lipid:siRNA~11:1 LNP092,2-Dilinoleyl-4- XTC/DSPC/Cholesterol/ dimethylaminoethyl- PEG-DMG[1,3]-dioxolane (XTC) 50/10/38.5/1.5 Lipid:siRNA 10:1 LNP10(3aR,5s,6aS)-N,N-dimethyl- ALN100/DSPC/Cholesterol/2,2-di((9Z,12Z)-octadeca- PEG-DMG 9,12-dienyl)tetrahydro- 50/10/38.5/1.53aH-cyclopenta[d] Lipid: siRNA 10:1 [1,3]dioxol-5-amine (ALN100) LNP11(6Z,9Z,28Z,31Z)- MC-3/DSPC/Cholesterol/ heptatriaconta- PEG-DMG6,9,28,31-tetraen- 50/10/38.5/1.5 19-yl 4-(dimethylamino) Lipid:siRNA10:1 butanoate (MC3) LNP12 1,1'-(2-(4-(2-((2-(bis(2- Tech G1/DSPC/hydroxydodecyl) Cholesterol/PEG-DMG amino)ethyl)(2- 50/10/38.5/1.5hydroxydodecyl)amino) Lipid:siRNA 10:1 ethyl)piperazin-1-yl)ethylazanediyl) didodecan-2-ol (Tech G1) LNP13 XTC XTC/DSPC/Chol/PEG-DMG 50/10/38.5/1.5 Lipid:siRNA: 33:1 LNP14 MC3 MC3/DSPC/Chol/PEG-DMG 40/15/40/5 Lipid:siRNA: 11:1 LNP15 MC3 MC3/DSPC/Chol/PEG-DSG/GalNAc- PEG-DSG/ 50/10/35/4.5/0.5 Lipid:siRNA: 11:1 LNP16 MC3MC3/DSPC/Chol/ PEG-DMG 50/10/38.5/1.5 Lipid:siRNA: 7:1 LNP17 MC3MC3/DSPC/Chol/ PEG-DSG 50/10/38.5/1.5 Lipid:siRNA: 10:1 LNP18 MC3MC3/DSPC/Chol/ PEG-DMG 50/10/38.5/1.5 Lipid:siRNA: 12:1 LNP19 MC3MC3/DSPC/Chol/ PEG-DMG 50/10/35/5 Lipid:siRNA: 8:1 LNP20 MC3MC3/DSPC/Chol/ PEG-DPG 50/10/38.5/1.5 Lipid:siRNA: 10:1 LNP21 C12-200C12-200/DSPC/Chol/ PEG-DSG 50/10/38.5/1.5 Lipid:siRNA: 7:1 LNP22 XTCXTC/DSPC/Chol/ PEG-DSG 50/10/38.5/1.5 Lipid:siRNA: 10:1 DSPC:distearoylphosphatidylcholine DPPC: dipalmitoylphosphatidylcholinePEG-DMG: PEG-didimyristoyl glycerol (C14-PEG, or PEG-C14) (PEG with avgmol wt of 2000) PEG-DSG: PEG-distyryl glycerol (C18-PEG, or PEG-C18)(PEG with avg mol wt of 2000) PEG-cDMA:PEG-carbamoyl-1,2-dimyristyloxypropylamine (PEG with avg mol wt of 2000)SNALP (1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLinDMA)) comprisingformulations are described in International PublicationNo.W02009/127060, filed Apr. 15, 2009, which is hereby incorporated byreference. XTC e.g., comprising formulations are described, in PCTPublication No. WO 2010/088537, the entire contents of which areincorporated herein by reference. MC3 comprising formulations aredescribed, e.g., in U.S. Publication No. 2010/0324120, filed Jun. 10,2010, the entire contents of which are incorporated herein by reference.ALNY-100 comprising formulations are described, e.g., PCT PublicationNo. WO 2010/054406, the entire contents of which are incorporated hereinby reference. C12-200 comprising formulations are described in PCTPublication No. WO 2010/129709, the entire contents of which areincorporated herein by reference.

Compositions and formulations for oral administration include powders orgranules, microparticulates, nanoparticulates, suspensions or solutionsin water or non-aqueous media, capsules, gel capsules, sachets, tabletsor minitablets. Thickeners, flavoring agents, diluents, emulsifiers,dispersing aids or binders can be desirable. In some embodiments, oralformulations are those in which dsRNAs featured in the invention areadministered in conjunction with one or more penetration enhancersurfactants and chelators. Suitable surfactants include fatty acidsand/or esters or salts thereof, bile acids and/or salts thereof.Suitable bile acids/salts include chenodeoxycholic acid (CDCA) andursodeoxychenodeoxycholic acid (UDCA), cholic acid, dehydrocholic acid,deoxycholic acid, glucholic acid, glycholic acid, glycodeoxycholic acid,taurocholic acid, taurodeoxycholic acid, sodiumtauro-24,25-dihydro-fusidate and sodium glycodihydrofusidate. Suitablefatty acids include arachidonic acid, undecanoic acid, oleic acid,lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid,stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate,monoolein, dilaurin, glyceryl 1-monocaprate,1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or amonoglyceride, a diglyceride or a pharmaceutically acceptable saltthereof (e.g., sodium). In some embodiments, combinations of penetrationenhancers are used, for example, fatty acids/salts in combination withbile acids/salts. One exemplary combination is the sodium salt of lauricacid, capric acid and UDCA. Further penetration enhancers includepolyoxyethylene-9-lauryl ether, polyoxyethylene-20-cetyl ether. DsRNAsfeatured in the invention can be delivered orally, in granular formincluding sprayed dried particles, or complexed to form micro ornanoparticles. DsRNA complexing agents include poly-amino acids;polyimines; polyacrylates; polyalkylacrylates, polyoxethanes,polyalkylcyanoacrylates; cationized gelatins, albumins, starches,acrylates, polyethyleneglycols (PEG) and starches;polyalkylcyanoacrylates; DEAE-derivatized polyimines, pollulans,celluloses and starches. Suitable complexing agents include chitosan,N-trimethylchitosan, poly-L-lysine, polyhistidine, polyornithine,polyspermines, protamine, polyvinylpyridine,polythiodiethylaminomethylethylene P(TDAE), polyaminostyrene (e.g.,p-amino), poly(methylcyanoacrylate), poly(ethylcyanoacrylate),poly(butylcyanoacrylate), poly(isobutylcyanoacrylate),poly(isohexylcynaoacrylate), DEAE-methacrylate, DEAE-hexylacrylate,DEAE-acrylamide, DEAE-albumin and DEAE-dextran, polymethylacrylate,polyhexylacrylate, poly(D,L-lactic acid), poly(DL-lactic-co-glycolicacid (PLGA), alginate, and polyethyleneglycol (PEG). Oral formulationsfor dsRNAs and their preparation are described in detail in U.S. Pat.No. 6,887,906, US Publn. No. 20030027780, and U.S. Pat. No. 6,747,014,each of which is incorporated herein by reference.

Compositions and formulations for parenteral, intraparenchymal (into thebrain), intrathecal, intraventricular or intrahepatic administration caninclude sterile aqueous solutions which can also contain buffers,diluents and other suitable additives such as, but not limited to,penetration enhancers, carrier compounds and other pharmaceuticallyacceptable carriers or excipients.

Pharmaceutical compositions of the present invention include, but arenot limited to, solutions, emulsions, and liposome-containingformulations. These compositions can be generated from a variety ofcomponents that include, but are not limited to, preformed liquids,self-emulsifying solids and self-emulsifying semisolids. Particularlypreferred are formulations that target the liver when treating hepaticdisorders such as hepatic carcinoma.

The pharmaceutical formulations of the present invention, which canconveniently be presented in unit dosage form, can be prepared accordingto conventional techniques well known in the pharmaceutical industry.Such techniques include the step of bringing into association the activeingredients with the pharmaceutical carrier(s) or excipient(s). Ingeneral, the formulations are prepared by uniformly and intimatelybringing into association the active ingredients with liquid carriers orfinely divided solid carriers or both, and then, if necessary, shapingthe product.

The compositions of the present invention can be formulated into any ofmany possible dosage forms such as, but not limited to, tablets,capsules, gel capsules, liquid syrups, soft gels, suppositories, andenemas. The compositions of the present invention can also be formulatedas suspensions in aqueous, non-aqueous or mixed media. Aqueoussuspensions can further contain substances which increase the viscosityof the suspension including, for example, sodium carboxymethylcellulose,sorbitol and/or dextran. The suspension can also contain stabilizers.

C. Additional Formulations

i. Emulsions

The compositions of the present invention can be prepared and formulatedas emulsions. Emulsions are typically heterogeneous systems of oneliquid dispersed in another in the form of droplets usually exceeding0.1 μm in diameter (see e.g., Ansel's Pharmaceutical Dosage Forms andDrug Delivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004,Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Idson, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199; Rosoff, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., Volume 1, p. 245; Block inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 2, p. 335; Higuchi et al.,in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton,Pa., 1985, p. 301). Emulsions are often biphasic systems comprising twoimmiscible liquid phases intimately mixed and dispersed with each other.In general, emulsions can be of either the water-in-oil (w/o) or theoil-in-water (o/w) variety. When an aqueous phase is finely divided intoand dispersed as minute droplets into a bulk oily phase, the resultingcomposition is called a water-in-oil (w/o) emulsion. Alternatively, whenan oily phase is finely divided into and dispersed as minute dropletsinto a bulk aqueous phase, the resulting composition is called anoil-in-water (o/w) emulsion. Emulsions can contain additional componentsin addition to the dispersed phases, and the active drug which can bepresent as a solution in either the aqueous phase, oily phase or itselfas a separate phase. Pharmaceutical excipients such as emulsifiers,stabilizers, dyes, and anti-oxidants can also be present in emulsions asneeded. Pharmaceutical emulsions can also be multiple emulsions that arecomprised of more than two phases such as, for example, in the case ofoil-in-water-in-oil (o/w/o) and water-in-oil-in-water (w/o/w) emulsions.Such complex formulations often provide certain advantages that simplebinary emulsions do not. Multiple emulsions in which individual oildroplets of an o/w emulsion enclose small water droplets constitute aw/o/w emulsion. Likewise a system of oil droplets enclosed in globulesof water stabilized in an oily continuous phase provides an o/w/oemulsion.

Emulsions are characterized by little or no thermodynamic stability.Often, the dispersed or discontinuous phase of the emulsion is welldispersed into the external or continuous phase and maintained in thisform through the means of emulsifiers or the viscosity of theformulation. Either of the phases of the emulsion can be a semisolid ora solid, as is the case of emulsion-style ointment bases and creams.Other means of stabilizing emulsions entail the use of emulsifiers thatcan be incorporated into either phase of the emulsion. Emulsifiers canbroadly be classified into four categories: synthetic surfactants,naturally occurring emulsifiers, absorption bases, and finely dispersedsolids (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug DeliverySystems, Allen, L V., Popovich N G., and Ansel H C., 2004, LippincottWilliams & Wilkins (8th ed.), New York, N.Y.; Idson, in PharmaceuticalDosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker,Inc., New York, N.Y., volume 1, p. 199).

Synthetic surfactants, also known as surface active agents, have foundwide applicability in the formulation of emulsions and have beenreviewed in the literature (see e.g., Ansel's Pharmaceutical DosageForms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel HC., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.;Rieger, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 285;Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), Marcel Dekker, Inc., New York, N.Y., 1988, volume 1, p. 199).Surfactants are typically amphiphilic and comprise a hydrophilic and ahydrophobic portion. The ratio of the hydrophilic to the hydrophobicnature of the surfactant has been termed the hydrophile/lipophilebalance (HLB) and is a valuable tool in categorizing and selectingsurfactants in the preparation of formulations. Surfactants can beclassified into different classes based on the nature of the hydrophilicgroup: nonionic, anionic, cationic and amphoteric (see e.g., Ansel'sPharmaceutical Dosage Forms and Drug Delivery Systems, Allen, L V.,Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8thed.), New York, N.Y. Rieger, in Pharmaceutical Dosage Forms, Lieberman,Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y.,volume 1, p. 285).

Naturally occurring emulsifiers used in emulsion formulations includelanolin, beeswax, phosphatides, lecithin and acacia. Absorption basespossess hydrophilic properties such that they can soak up water to formw/o emulsions yet retain their semisolid consistencies, such asanhydrous lanolin and hydrophilic petrolatum. Finely divided solids havealso been used as good emulsifiers especially in combination withsurfactants and in viscous preparations. These include polar inorganicsolids, such as heavy metal hydroxides, nonswelling clays such asbentonite, attapulgite, hectorite, kaolin, montmorillonite, colloidalaluminum silicate and colloidal magnesium aluminum silicate, pigmentsand nonpolar solids such as carbon or glyceryl tristearate.

A large variety of non-emulsifying materials are also included inemulsion formulations and contribute to the properties of emulsions.These include fats, oils, waxes, fatty acids, fatty alcohols, fattyesters, humectants, hydrophilic colloids, preservatives and antioxidants(Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335;Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).

Hydrophilic colloids or hydrocolloids include naturally occurring gumsand synthetic polymers such as polysaccharides (for example, acacia,agar, alginic acid, carrageenan, guar gum, karaya gum, and tragacanth),cellulose derivatives (for example, carboxymethylcellulose andcarboxypropylcellulose), and synthetic polymers (for example, carbomers,cellulose ethers, and carboxyvinyl polymers). These disperse or swell inwater to form colloidal solutions that stabilize emulsions by formingstrong interfacial films around the dispersed-phase droplets and byincreasing the viscosity of the external phase.

Since emulsions often contain a number of ingredients such ascarbohydrates, proteins, sterols and phosphatides that can readilysupport the growth of microbes, these formulations often incorporatepreservatives. Commonly used preservatives included in emulsionformulations include methyl paraben, propyl paraben, quaternary ammoniumsalts, benzalkonium chloride, esters of p-hydroxybenzoic acid, and boricacid. Antioxidants are also commonly added to emulsion formulations toprevent deterioration of the formulation. Antioxidants used can be freeradical scavengers such as tocopherols, alkyl gallates, butylatedhydroxyanisole, butylated hydroxytoluene, or reducing agents such asascorbic acid and sodium metabisulfite, and antioxidant synergists suchas citric acid, tartaric acid, and lecithin.

The application of emulsion formulations via dermatological, oral andparenteral routes and methods for their manufacture have been reviewedin the literature (see e.g., Ansel's Pharmaceutical Dosage Forms andDrug Delivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004,Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Idson, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199). Emulsionformulations for oral delivery have been very widely used because ofease of formulation, as well as efficacy from an absorption andbioavailability standpoint (see e.g., Ansel's Pharmaceutical DosageForms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel HC., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.;Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245;Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).Mineral-oil base laxatives, oil-soluble vitamins and high fat nutritivepreparations are among the materials that have commonly beenadministered orally as o/w emulsions.

ii. Microemulsions

In one embodiment of the present invention, the compositions of iRNAsand nucleic acids are formulated as microemulsions. A microemulsion canbe defined as a system of water, oil and amphiphile which is a singleoptically isotropic and thermodynamically stable liquid solution (seee.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems,Allen, L V., Popovich N G., and Ansel H C., 2004, Lippincott Williams &Wilkins (8th ed.), New York, N.Y.; Rosoff, in Pharmaceutical DosageForms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc.,New York, N.Y., volume 1, p. 245). Typically microemulsions are systemsthat are prepared by first dispersing an oil in an aqueous surfactantsolution and then adding a sufficient amount of a fourth component,generally an intermediate chain-length alcohol to form a transparentsystem. Therefore, microemulsions have also been described asthermodynamically stable, isotropically clear dispersions of twoimmiscible liquids that are stabilized by interfacial films ofsurface-active molecules (Leung and Shah, in: Controlled Release ofDrugs: Polymers and Aggregate Systems, Rosoff, M., Ed., 1989, VCHPublishers, New York, pages 185-215). Microemulsions commonly areprepared via a combination of three to five components that include oil,water, surfactant, cosurfactant and electrolyte. Whether themicroemulsion is of the water-in-oil (w/o) or an oil-in-water (o/w) typeis dependent on the properties of the oil and surfactant used and on thestructure and geometric packing of the polar heads and hydrocarbon tailsof the surfactant molecules (Schott, in Remington's PharmaceuticalSciences, Mack Publishing Co., Easton, Pa., 1985, p. 271).

The phenomenological approach utilizing phase diagrams has beenextensively studied and has yielded a comprehensive knowledge, to oneskilled in the art, of how to formulate microemulsions (see e.g.,Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins(8th ed.), New York, N.Y.; Rosoff, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., NewYork, N.Y., volume 1, p. 245; Block, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., NewYork, N.Y., volume 1, p. 335). Compared to conventional emulsions,microemulsions offer the advantage of solubilizing water-insoluble drugsin a formulation of thermodynamically stable droplets that are formedspontaneously.

Surfactants used in the preparation of microemulsions include, but arenot limited to, ionic surfactants, non-ionic surfactants, Brij 96,polyoxyethylene oleyl ethers, polyglycerol fatty acid esters,tetraglycerol monolaurate (ML310), tetraglycerol monooleate (MO310),hexaglycerol monooleate (PO310), hexaglycerol pentaoleate (PO500),decaglycerol monocaprate (MCA750), decaglycerol monooleate (MO750),decaglycerol sequioleate (SO750), decaglycerol decaoleate (DAO750),alone or in combination with cosurfactants. The cosurfactant, usually ashort-chain alcohol such as ethanol, 1-propanol, and 1-butanol, servesto increase the interfacial fluidity by penetrating into the surfactantfilm and consequently creating a disordered film because of the voidspace generated among surfactant molecules. Microemulsions can, however,be prepared without the use of cosurfactants and alcohol-freeself-emulsifying microemulsion systems are known in the art. The aqueousphase can typically be, but is not limited to, water, an aqueoussolution of the drug, glycerol, PEG300, PEG400, polyglycerols, propyleneglycols, and derivatives of ethylene glycol. The oil phase can include,but is not limited to, materials such as Captex 300, Captex 355, CapmulMCM, fatty acid esters, medium chain (C8-C12) mono, di, andtri-glycerides, polyoxyethylated glyceryl fatty acid esters, fattyalcohols, polyglycolized glycerides, saturated polyglycolized C8-C10glycerides, vegetable oils and silicone oil.

Microemulsions are particularly of interest from the standpoint of drugsolubilization and the enhanced absorption of drugs. Lipid basedmicroemulsions (both o/w and w/o) have been proposed to enhance the oralbioavailability of drugs, including peptides (see e.g., U.S. Pat. Nos.6,191,105; 7,063,860; 7,070,802; 7,157,099; Constantinides et al.,Pharmaceutical Research, 1994, 11, 1385-1390; Ritschel, Meth. Find. Exp.Clin. Pharmacol., 1993, 13, 205). Microemulsions afford advantages ofimproved drug solubilization, protection of drug from enzymatichydrolysis, possible enhancement of drug absorption due tosurfactant-induced alterations in membrane fluidity and permeability,ease of preparation, ease of oral administration over solid dosageforms, improved clinical potency, and decreased toxicity (see e.g., U.S.Pat. Nos. 6,191,105; 7,063,860; 7,070,802; 7,157,099; Constantinides etal., Pharmaceutical Research, 1994, 11, 1385; Ho et al., J. Pharm. Sci.,1996, 85, 138-143). Often microemulsions can form spontaneously whentheir components are brought together at ambient temperature. This canbe particularly advantageous when formulating thermolabile drugs,peptides or iRNAs. Microemulsions have also been effective in thetransdermal delivery of active components in both cosmetic andpharmaceutical applications. It is expected that the microemulsioncompositions and formulations of the present invention will facilitatethe increased systemic absorption of iRNAs and nucleic acids from thegastrointestinal tract, as well as improve the local cellular uptake ofiRNAs and nucleic acids.

Microemulsions of the present invention can also contain additionalcomponents and additives such as sorbitan monostearate (Grill 3),Labrasol, and penetration enhancers to improve the properties of theformulation and to enhance the absorption of the iRNAs and nucleic acidsof the present invention. Penetration enhancers used in themicroemulsions of the present invention can be classified as belongingto one of five broad categories—surfactants, fatty acids, bile salts,chelating agents, and non-chelating non-surfactants (Lee et al.,Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92). Eachof these classes has been discussed above.

iii. Microparticles

An iRNA agent of the invention may be incorporated into a particle,e.g., a microparticle. Microparticles can be produced by spray-drying,but may also be produced by other methods including lyophilization,evaporation, fluid bed drying, vacuum drying, or a combination of thesetechniques.

iv. Penetration Enhancers

In one embodiment, the present invention employs various penetrationenhancers to effect the efficient delivery of nucleic acids,particularly iRNAs, to the skin of animals. Most drugs are present insolution in both ionized and nonionized forms. However, usually onlylipid soluble or lipophilic drugs readily cross cell membranes. It hasbeen discovered that even non-lipophilic drugs can cross cell membranesif the membrane to be crossed is treated with a penetration enhancer. Inaddition to aiding the diffusion of non-lipophilic drugs across cellmembranes, penetration enhancers also enhance the permeability oflipophilic drugs.

Penetration enhancers can be classified as belonging to one of fivebroad categories, i.e., surfactants, fatty acids, bile salts, chelatingagents, and non-chelating non-surfactants (see e.g., Malmsten, M.Surfactants and polymers in drug delivery, Informa Health Care, NewYork, N.Y., 2002; Lee et al., Critical Reviews in Therapeutic DrugCarrier Systems, 1991, p. 92). Each of the above mentioned classes ofpenetration enhancers are described below in greater detail.

Surfactants (or “surface-active agents”) are chemical entities which,when dissolved in an aqueous solution, reduce the surface tension of thesolution or the interfacial tension between the aqueous solution andanother liquid, with the result that absorption of iRNAs through themucosa is enhanced. In addition to bile salts and fatty acids, thesepenetration enhancers include, for example, sodium lauryl sulfate,polyoxyethylene-9-lauryl ether and polyoxyethylene-20-cetyl ether) (seee.g., Malmsten, M. Surfactants and polymers in drug delivery, InformaHealth Care, New York, N.Y., 2002; Lee et al., Critical Reviews inTherapeutic Drug Carrier Systems, 1991, p. 92); and perfluorochemicalemulsions, such as FC-43. Takahashi et al., J. Pharm. Pharmacol., 1988,40, 252).

Various fatty acids and their derivatives which act as penetrationenhancers include, for example, oleic acid, lauric acid, capric acid(n-decanoic acid), myristic acid, palmitic acid, stearic acid, linoleicacid, linolenic acid, dicaprate, tricaprate, monoolein(1-monooleoyl-rac-glycerol), dilaurin, caprylic acid, arachidonic acid,glycerol 1-monocaprate, 1-dodecylazacycloheptan-2-one, acylcarnitines,acylcholines, C1-20 alkyl esters thereof (e.g., methyl, isopropyl andt-butyl), and mono- and di-glycerides thereof (i.e., oleate, laurate,caprate, myristate, palmitate, stearate, linoleate, etc.) (see e.g.,Touitou, E., et al. Enhancement in Drug Delivery, CRC Press, Danvers,Mass., 2006; Lee et al., Critical Reviews in Therapeutic Drug CarrierSystems, 1991, p. 92; Muranishi, Critical Reviews in Therapeutic DrugCarrier Systems, 1990, 7, 1-33; El Hariri et al., J. Pharm. Pharmacol.,1992, 44, 651-654).

The physiological role of bile includes the facilitation of dispersionand absorption of lipids and fat-soluble vitamins (see e.g., Malmsten,M. Surfactants and polymers in drug delivery, Informa Health Care, NewYork, N.Y., 2002; Brunton, Chapter 38 in: Goodman & Gilman's ThePharmacological Basis of Therapeutics, 9th Ed., Hardman et al. Eds.,McGraw-Hill, New York, 1996, pp. 934-935). Various natural bile salts,and their synthetic derivatives, act as penetration enhancers. Thus theterm “bile salts” includes any of the naturally occurring components ofbile as well as any of their synthetic derivatives. Suitable bile saltsinclude, for example, cholic acid (or its pharmaceutically acceptablesodium salt, sodium cholate), dehydrocholic acid (sodiumdehydrocholate), deoxycholic acid (sodium deoxycholate), glucholic acid(sodium glucholate), glycholic acid (sodium glycocholate),glycodeoxycholic acid (sodium glycodeoxycholate), taurocholic acid(sodium taurocholate), taurodeoxycholic acid (sodium taurodeoxycholate),chenodeoxycholic acid (sodium chenodeoxycholate), ursodeoxycholic acid(UDCA), sodium tauro-24,25-dihydro-fusidate (STDHF), sodiumglycodihydrofusidate and polyoxyethylene-9-lauryl ether (POE) (see e.g.,Malmsten, M. Surfactants and polymers in drug delivery, Informa HealthCare, New York, N.Y., 2002; Lee et al., Critical Reviews in TherapeuticDrug Carrier Systems, 1991, page 92; Swinyard, Chapter 39 In:Remington's Pharmaceutical Sciences, 18th Ed., Gennaro, ed., MackPublishing Co., Easton, Pa., 1990, pages 782-783; Muranishi, CriticalReviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; Yamamoto etal., J. Pharm. Exp. Ther., 1992, 263, 25; Yamashita et al., J. Pharm.Sci., 1990, 79, 579-583).

Chelating agents, as used in connection with the present invention, canbe defined as compounds that remove metallic ions from solution byforming complexes therewith, with the result that absorption of iRNAsthrough the mucosa is enhanced. With regards to their use as penetrationenhancers in the present invention, chelating agents have the addedadvantage of also serving as DNase inhibitors, as most characterized DNAnucleases require a divalent metal ion for catalysis and are thusinhibited by chelating agents (Jarrett, J. Chromatogr., 1993, 618,315-339). Suitable chelating agents include but are not limited todisodium ethylenediaminetetraacetate (EDTA), citric acid, salicylates(e.g., sodium salicylate, 5-methoxysalicylate and homovanilate), N-acylderivatives of collagen, laureth-9 and N-amino acyl derivatives ofbeta-diketones (enamines)(see e.g., Katdare, A. et al., Excipientdevelopment for pharmaceutical, biotechnology, and drug delivery, CRCPress, Danvers, Mass., 2006; Lee et al., Critical Reviews in TherapeuticDrug Carrier Systems, 1991, page 92; Muranishi, Critical Reviews inTherapeutic Drug Carrier Systems, 1990, 7, 1-33; Buur et al., J. ControlRel., 1990, 14, 43-51).

As used herein, non-chelating non-surfactant penetration enhancingcompounds can be defined as compounds that demonstrate insignificantactivity as chelating agents or as surfactants but that nonethelessenhance absorption of iRNAs through the alimentary mucosa (see e.g.,Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990,7, 1-33). This class of penetration enhancers includes, for example,unsaturated cyclic ureas, 1-alkyl- and 1-alkenylazacyclo-alkanonederivatives (Lee et al., Critical Reviews in Therapeutic Drug CarrierSystems, 1991, page 92); and non-steroidal anti-inflammatory agents suchas diclofenac sodium, indomethacin and phenylbutazone (Yamashita et al.,J. Pharm. Pharmacol., 1987, 39, 621-626).

Agents that enhance uptake of iRNAs at the cellular level can also beadded to the pharmaceutical and other compositions of the presentinvention. For example, cationic lipids, such as lipofectin (Junichi etal, U.S. Pat. No. 5,705,188), cationic glycerol derivatives, andpolycationic molecules, such as polylysine (Lollo et al., PCTApplication WO 97/30731), are also known to enhance the cellular uptakeof dsRNAs. Examples of commercially available transfection reagentsinclude, for example Lipofectamine™ (Invitrogen; Carlsbad, Calif.),Lipofectamine 2000™ (Invitrogen; Carlsbad, Calif.), 293Fectin™(Invitrogen; Carlsbad, Calif.), Cellfectin™ (Invitrogen; Carlsbad,Calif.), DMRIE-C™ (Invitrogen; Carlsbad, Calif.), FreeStyle™ MAX(Invitrogen; Carlsbad, Calif.), Lipofectamine™ 2000 CD (Invitrogen;Carlsbad, Calif.), Lipofectamine™ (Invitrogen; Carlsbad, Calif.),iRNAMAX (Invitrogen; Carlsbad, Calif.), Oligofectamine™ (Invitrogen;Carlsbad, Calif.), Optifect™ (Invitrogen; Carlsbad, Calif.), X-tremeGENEQ2 Transfection Reagent (Roche; Grenzacherstrasse, Switzerland), DOTAPLiposomal Transfection Reagent (Grenzacherstrasse, Switzerland), DOSPERLiposomal Transfection Reagent (Grenzacherstrasse, Switzerland), orFugene (Grenzacherstrasse, Switzerland), Transfectam® Reagent (Promega;Madison, Wis.), TransFast™ Transfection Reagent (Promega; Madison,Wis.), Tfx™-20 Reagent (Promega; Madison, Wis.), Tfx™-50 Reagent(Promega; Madison, Wis.), DreamFect™ (OZ Biosciences; Marseille,France), EcoTransfect (OZ Biosciences; Marseille, France), TransPass^(a)D1 Transfection Reagent (New England Biolabs; Ipswich, Mass., USA),LyoVec™/LipoGen™ (Invitrogen; San Diego, Calif., USA), PerFectinTransfection Reagent (Genlantis; San Diego, Calif., USA), NeuroPORTERTransfection Reagent (Genlantis; San Diego, Calif., USA), GenePORTERTransfection reagent (Genlantis; San Diego, Calif., USA), GenePORTER 2Transfection reagent (Genlantis; San Diego, Calif., USA), CytofectinTransfection Reagent (Genlantis; San Diego, Calif., USA), BaculoPORTERTransfection Reagent (Genlantis; San Diego, Calif., USA), TroganPORTER™transfection Reagent (Genlantis; San Diego, Calif., USA), RiboFect(Bioline; Taunton, Mass., USA), PlasFect (Bioline; Taunton, Mass., USA),UniFECTOR (B-Bridge International; Mountain View, Calif., USA),SureFECTOR (B-Bridge International; Mountain View, Calif., USA), orHiFect™ (B-Bridge International, Mountain View, Calif., USA), amongothers.

Other agents can be utilized to enhance the penetration of theadministered nucleic acids, including glycols such as ethylene glycoland propylene glycol, pyrrols such as 2-pyrrol, azones, and terpenessuch as limonene and menthone.

v. Carriers

Certain compositions of the present invention also incorporate carriercompounds in the formulation. As used herein, “carrier compound” or“carrier” can refer to a nucleic acid, or analog thereof, which is inert(i.e., does not possess biological activity per se) but is recognized asa nucleic acid by in vivo processes that reduce the bioavailability of anucleic acid having biological activity by, for example, degrading thebiologically active nucleic acid or promoting its removal fromcirculation. The coadministration of a nucleic acid and a carriercompound, typically with an excess of the latter substance, can resultin a substantial reduction of the amount of nucleic acid recovered inthe liver, kidney or other extracirculatory reservoirs, presumably dueto competition between the carrier compound and the nucleic acid for acommon receptor. For example, the recovery of a partiallyphosphorothioate dsRNA in hepatic tissue can be reduced when it iscoadministered with polyinosinic acid, dextran sulfate, polycytidic acidor 4-acetamido-4′isothiocyano-stilbene-2,2′-disulfonic acid (Miyao etal., DsRNA Res. Dev., 1995, 5, 115-121; Takakura et al., DsRNA & Nucl.Acid Drug Dev., 1996, 6, 177-183.

vi. Excipients

In contrast to a carrier compound, a “pharmaceutical carrier” or“excipient” is a pharmaceutically acceptable solvent, suspending agentor any other pharmacologically inert vehicle for delivering one or morenucleic acids to an animal. The excipient can be liquid or solid and isselected, with the planned manner of administration in mind, so as toprovide for the desired bulk, consistency, etc., when combined with anucleic acid and the other components of a given pharmaceuticalcomposition. Typical pharmaceutical carriers include, but are notlimited to, binding agents (e.g., pregelatinized maize starch,polyvinylpyrrolidone or hydroxypropyl methylcellulose, etc.); fillers(e.g., lactose and other sugars, microcrystalline cellulose, pectin,gelatin, calcium sulfate, ethyl cellulose, polyacrylates or calciumhydrogen phosphate, etc.); lubricants (e.g., magnesium stearate, talc,silica, colloidal silicon dioxide, stearic acid, metallic stearates,hydrogenated vegetable oils, corn starch, polyethylene glycols, sodiumbenzoate, sodium acetate, etc.); disintegrants (e.g., starch, sodiumstarch glycolate, etc.); and wetting agents (e.g., sodium laurylsulphate, etc).

Pharmaceutically acceptable organic or inorganic excipients suitable fornon-parenteral administration which do not deleteriously react withnucleic acids can also be used to formulate the compositions of thepresent invention. Suitable pharmaceutically acceptable carriersinclude, but are not limited to, water, salt solutions, alcohols,polyethylene glycols, gelatin, lactose, amylose, magnesium stearate,talc, silicic acid, viscous paraffin, hydroxymethylcellulose,polyvinylpyrrolidone and the like.

Formulations for topical administration of nucleic acids can includesterile and non-sterile aqueous solutions, non-aqueous solutions incommon solvents such as alcohols, or solutions of the nucleic acids inliquid or solid oil bases. The solutions can also contain buffers,diluents and other suitable additives. Pharmaceutically acceptableorganic or inorganic excipients suitable for non-parenteraladministration which do not deleteriously react with nucleic acids canbe used.

Suitable pharmaceutically acceptable excipients include, but are notlimited to, water, salt solutions, alcohol, polyethylene glycols,gelatin, lactose, amylose, magnesium stearate, talc, silicic acid,viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and thelike.

vii. Other Components

The compositions of the present invention can additionally contain otheradjunct components conventionally found in pharmaceutical compositions,at their art-established usage levels. Thus, for example, thecompositions can contain additional, compatible, pharmaceutically-activematerials such as, for example, antipruritics, astringents, localanesthetics or anti-inflammatory agents, or can contain additionalmaterials useful in physically formulating various dosage forms of thecompositions of the present invention, such as dyes, flavoring agents,preservatives, antioxidants, opacifiers, thickening agents andstabilizers. However, such materials, when added, should not undulyinterfere with the biological activities of the components of thecompositions of the present invention. The formulations can besterilized and, if desired, mixed with auxiliary agents, e.g.,lubricants, preservatives, stabilizers, wetting agents, emulsifiers,salts for influencing osmotic pressure, buffers, colorings, flavoringsand/or aromatic substances and the like which do not deleteriouslyinteract with the nucleic acid(s) of the formulation.

Aqueous suspensions can contain substances which increase the viscosityof the suspension including, for example, sodium carboxymethylcellulose,sorbitol and/or dextran. The suspension can also contain stabilizers.

In some embodiments, pharmaceutical compositions featured in theinvention include (a) one or more iRNA compounds and (b) one or moreagents which function by a non-iRNA mechanism and which are useful intreating an HDV infection. Examples of such agents include, but are notlimited to antiviral agents aimed at suppressing or destroying HDVand/or HBV by interfering with viral replication; and immune modulatorsaimed at helping the human immune system mount a defense against thevirus. In contrast, immune modulators, such as corticosteroids, whichinduce an enhanced expression of virus and viral antigens, and asuppression of T-lymphocyte function, or adenine arabinoside, acyclovir,or dideoxyinosine, are not beneficial for the treatment of chronichepatitis B and/or chronic hepatitis D. Suitable agents are discussed inmore detail below.

Toxicity and therapeutic efficacy of such compounds can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the LD50 (the dose lethal to 50% of thepopulation) and the ED50 (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio LD50/ED50.Compounds that exhibit high therapeutic indices are preferred.

The data obtained from cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofcompositions featured herein in the invention lies generally within arange of circulating concentrations that include the ED50 with little orno toxicity. The dosage can vary within this range depending upon thedosage form employed and the route of administration utilized. For anycompound used in the methods featured in the invention, thetherapeutically effective dose can be estimated initially from cellculture assays. A dose can be formulated in animal models to achieve acirculating plasma concentration range of the compound or, whenappropriate, of the polypeptide product of a target sequence (e.g.,achieving a decreased concentration of the polypeptide) that includesthe IC50 (i.e., the concentration of the test compound which achieves ahalf-maximal inhibition of symptoms) as determined in cell culture. Suchinformation can be used to more accurately determine useful doses inhumans. Levels in plasma can be measured, for example, by highperformance liquid chromatography.

In addition to their administration, as discussed above, the iRNAsfeatured in the invention can be administered in combination with otherknown agents effective in treatment of pathological processes mediatedby HDV expression. In any event, the administering physician can adjustthe amount and timing of iRNA administration on the basis of resultsobserved using standard measures of efficacy known in the art ordescribed herein.

VII. Methods of the Invention

The present invention provides therapeutic and prophylactic methodswhich include administering to a subject having an HDV infection and/orHDV-associated disease, disorder, and/or condition, or prone todeveloping, an HDV-associated disease, disorder, and/or condition (e.g.,CHB), compositions comprising an iRNA agent, or pharmaceuticalcompositions comprising an iRNA agent, or vectors comprising an iRNA ofthe invention.

The methods of the invention are useful for treating a subject having anHDV infection, e.g., a subject that would benefit from reduction in HDVgene expression and/or HDV replication. In one aspect, the presentinvention provides methods of reducing the level of Hepatis B virus cccDNA in a subject infected with HDV. In another aspect, the presentinvention provides methods of reducing the level of HDV antigen, e.g.,HBsAg and/or HBeAg, in a subject infected with HDV. In another aspect,the present invention provides methods of reducing the viral load of HDVin a subject infected with HDV. The present invention also providesmethods of reducing the level of alanine aminotransferase (ALT) and/oraspartate aminotransferase (AST) in a subject infected with HDV. In oneaspect, the present invention provides methods for increasing the levelof anti-HDV antibodies in a subject infected with HDV. In anotheraspect, the present invention provides methods of treating a subjecthaving an HDV infection. In one aspect, the present invention providesmethods of treating a subject having an HDV-associated disease, e.g.,acute hepatitis D; hepatitis B virus infection, acute hepatitis B, acutefulminant hepatitis D; chronic hepatitis D; liver fibrosis; end-stageliver disease; hepatocellular carcinoma. The treatment methods (anduses) of the invention include administering to the subject, e.g., ahuman, a therapeutically effective amount of an iRNA agent of theinvention targeting an HDV gene or a pharmaceutical compositioncomprising an iRNA agent of the invention targeting an HDV gene or avector of the invention comprising an iRNA agent targeting an HDV gene.

In one aspect, the invention provides methods of preventing at least onesymptom in a subject having an HDV infection,

In another aspect, the present invention provides uses of atherapeutically effective amount of an iRNA agent of the invention fortreating a subject, e.g., a subject that would benefit from a reductionand/or inhibition of HDV gene expression.

In a further aspect, the present invention provides uses of an iRNAagent, e.g., a dsRNA, of the invention targeting an HDV gene orpharmaceutical composition comprising an iRNA agent targeting an HDVgene in the manufacture of a medicament for treating a subject, e.g., asubject that would benefit from a reduction and/or inhibition of HDVgene expression and/or HDV replication, such as a subject having adisorder that would benefit from reduction in HDV gene expression, e.g.,a HDV-associated disease.

In another aspect, the invention provides uses of an iRNA, e.g., adsRNA, of the invention for preventing at least one symptom in a subjectsuffering from a disorder that would benefit from a reduction and/orinhibition of HDV gene expression and/or HDV replication.

In a further aspect, the present invention provides uses of an iRNAagent of the invention in the manufacture of a medicament for preventingat least one symptom in a subject suffering from a disorder that wouldbenefit from a reduction and/or inhibition of HDV gene expression and/orHDV replication, such as a HDV-associated disease.

In one embodiment, an iRNA agent targeting HDV is administered to asubject having an HDV infection and/or an HDV-associated disease suchthat the expression of one or more HDV genes, HDV antigen levels, and/orHDV viral load levels, e.g., in a cell, tissue, blood or other tissue orfluid of the subject are reduced by at least about 10%, 11%, 12%, 13%,14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%,28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%,42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%,56%, 57%, 58%, 59%, 60%, 61%, 62%, 62%, 64%, 65%, 66%, 67%, 68%, 69%,70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or at least about 99% or more when the dsRNA agent is administeredto the subject.

In one embodiment, an iRNA agent targeting HDV is administered to asubject having an HDV infection and/or an HDV-associated disease suchthat the level of anti-HDV antibodies, e.g., in a cell, tissue, blood orother tissue or fluid of the subject are increased by at least about10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%,24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%,38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%,52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 62%, 64%, 65%,66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%,80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, or at least about 99% or more when the dsRNAagent is administered to the subject.

The methods and uses of the invention include administering acomposition described herein such that expression of the target HDV geneis decreased, such as for about 1, 2, 3, 4 5, 6, 7, 8, 12, 16, 18, 24,28, 32, 36, 40, 44, 48, 52, 56, 60, 64, 68, 72, 76, or about 80 hours.In one embodiment, expression of the target HDV gene is decreased for anextended duration, e.g., at least about two, three, four, five, six,seven days or more, e.g., about one week, two weeks, three weeks, orabout four weeks or longer.

Administration of the dsRNA according to the methods and uses of theinvention may result in a reduction of the severity, signs, symptoms,and/or markers of such diseases or disorders in a patient with an HDVinfection and/or HDV-associated disease. By “reduction” in this contextis meant a statistically significant decrease in such level. Thereduction can be, for example, at least about 5%, 10%, 15%, 20%, 25%,30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, orabout 100%.

Efficacy of treatment or prevention of disease can be assessed, forexample by measuring disease progression, disease remission, symptomseverity, reduction in pain, quality of life, dose of a medicationrequired to sustain a treatment effect, level of a disease marker or anyother measurable parameter appropriate for a given disease being treatedor targeted for prevention. It is well within the ability of one skilledin the art to monitor efficacy of treatment or prevention by measuringany one of such parameters, or any combination of parameters. Forexample, efficacy of treatment of CHB may be assessed, for example, byperiodic monitoring of viral load and transaminase levels. Comparison ofthe later readings with the initial readings provide a physician anindication of whether the treatment is effective. It is well within theability of one skilled in the art to monitor efficacy of treatment orprevention by measuring any one of such parameters, or any combinationof parameters. In connection with the administration of an iRNAtargeting HDV or pharmaceutical composition thereof, “effective against”an HDV-associated disease indicates that administration in a clinicallyappropriate manner results in a beneficial effect for at least astatistically significant fraction of patients, such as improvement ofsymptoms, a cure, a reduction in disease, extension of life, improvementin quality of life, or other effect generally recognized as positive bymedical doctors familiar with treating HDV infection and/or anHDV-associated disease and the related causes.

A treatment or preventive effect is evident when there is astatistically significant improvement in one or more parameters ofdisease status, or by a failure to worsen or to develop symptoms wherethey would otherwise be anticipated. As an example, a favorable changeof at least 10% in a measurable parameter of disease, and preferably atleast 20%, 30%, 40%, 50% or more can be indicative of effectivetreatment. Efficacy for a given iRNA drug or formulation of that drugcan also be judged using an experimental animal model for the givendisease as known in the art. When using an experimental animal model,efficacy of treatment is evidenced when a statistically significantreduction in a marker or symptom is observed.

Subjects can be administered a therapeutic amount of iRNA, such as about0.01 mg/kg, 0.02 mg/kg, 0.03 mg/kg, 0.04 mg/kg, 0.05 mg/kg, 0.1 mg/kg,0.15 mg/kg, 0.2 mg/kg, 0.25 mg/kg, 0.3 mg/kg, 0.35 mg/kg, 0.4 mg/kg,0.45 mg/kg, 0.5 mg/kg, 0.55 mg/kg, 0.6 mg/kg, 0.65 mg/kg, 0.7 mg/kg,0.75 mg/kg, 0.8 mg/kg, 0.85 mg/kg, 0.9 mg/kg, 0.95 mg/kg, 1.0 mg/kg, 1.1mg/kg, 1.2 mg/kg, 1.3 mg/kg, 1.4 mg/kg, 1.5 mg/kg, 1.6 mg/kg, 1.7 mg/kg,1.8 mg/kg, 1.9 mg/kg, 2.0 mg/kg, 2.1 mg/kg, 2.2 mg/kg, 2.3 mg/kg, 2.4mg/kg, 2.5 mg/kg dsRNA, 2.6 mg/kg dsRNA, 2.7 mg/kg dsRNA, 2.8 mg/kgdsRNA, 2.9 mg/kg dsRNA, 3.0 mg/kg dsRNA, 3.1 mg/kg dsRNA, 3.2 mg/kgdsRNA, 3.3 mg/kg dsRNA, 3.4 mg/kg dsRNA, 3.5 mg/kg dsRNA, 3.6 mg/kgdsRNA, 3.7 mg/kg dsRNA, 3.8 mg/kg dsRNA, 3.9 mg/kg dsRNA, 4.0 mg/kgdsRNA, 4.1 mg/kg dsRNA, 4.2 mg/kg dsRNA, 4.3 mg/kg dsRNA, 4.4 mg/kgdsRNA, 4.5 mg/kg dsRNA, 4.6 mg/kg dsRNA, 4.7 mg/kg dsRNA, 4.8 mg/kgdsRNA, 4.9 mg/kg dsRNA, 5.0 mg/kg dsRNA, 5.1 mg/kg dsRNA, 5.2 mg/kgdsRNA, 5.3 mg/kg dsRNA, 5.4 mg/kg dsRNA, 5.5 mg/kg dsRNA, 5.6 mg/kgdsRNA, 5.7 mg/kg dsRNA, 5.8 mg/kg dsRNA, 5.9 mg/kg dsRNA, 6.0 mg/kgdsRNA, 6.1 mg/kg dsRNA, 6.2 mg/kg dsRNA, 6.3 mg/kg dsRNA, 6.4 mg/kgdsRNA, 6.5 mg/kg dsRNA, 6.6 mg/kg dsRNA, 6.7 mg/kg dsRNA, 6.8 mg/kgdsRNA, 6.9 mg/kg dsRNA, 7.0 mg/kg dsRNA, 7.1 mg/kg dsRNA, 7.2 mg/kgdsRNA, 7.3 mg/kg dsRNA, 7.4 mg/kg dsRNA, 7.5 mg/kg dsRNA, 7.6 mg/kgdsRNA, 7.7 mg/kg dsRNA, 7.8 mg/kg dsRNA, 7.9 mg/kg dsRNA, 8.0 mg/kgdsRNA, 8.1 mg/kg dsRNA, 8.2 mg/kg dsRNA, 8.3 mg/kg dsRNA, 8.4 mg/kgdsRNA, 8.5 mg/kg dsRNA, 8.6 mg/kg dsRNA, 8.7 mg/kg dsRNA, 8.8 mg/kgdsRNA, 8.9 mg/kg dsRNA, 9.0 mg/kg dsRNA, 9.1 mg/kg dsRNA, 9.2 mg/kgdsRNA, 9.3 mg/kg dsRNA, 9.4 mg/kg dsRNA, 9.5 mg/kg dsRNA, 9.6 mg/kgdsRNA, 9.7 mg/kg dsRNA, 9.8 mg/kg dsRNA, 9.9 mg/kg dsRNA, 9.0 mg/kgdsRNA, 10 mg/kg dsRNA, 15 mg/kg dsRNA, 20 mg/kg dsRNA, 25 mg/kg dsRNA,30 mg/kg dsRNA, 35 mg/kg dsRNA, 40 mg/kg dsRNA, 45 mg/kg dsRNA, or about50 mg/kg dsRNA. Values and ranges intermediate to the recited values arealso intended to be part of this invention.

In certain embodiments, for example, when a composition of the inventioncomprises a dsRNA as described herein and a lipid, subjects can beadministered a therapeutic amount of iRNA, such as about 0.01 mg/kg toabout 5 mg/kg, about 0.01 mg/kg to about 10 mg/kg, about 0.05 mg/kg toabout 5 mg/kg, about 0.05 mg/kg to about 10 mg/kg, about 0.1 mg/kg toabout 5 mg/kg, about 0.1 mg/kg to about 10 mg/kg, about 0.2 mg/kg toabout 5 mg/kg, about 0.2 mg/kg to about 10 mg/kg, about 0.3 mg/kg toabout 5 mg/kg, about 0.3 mg/kg to about 10 mg/kg, about 0.4 mg/kg toabout 5 mg/kg, about 0.4 mg/kg to about 10 mg/kg, about 0.5 mg/kg toabout 5 mg/kg, about 0.5 mg/kg to about 10 mg/kg, about 1 mg/kg to about5 mg/kg, about 1 mg/kg to about 10 mg/kg, about 1.5 mg/kg to about 5mg/kg, about 1.5 mg/kg to about 10 mg/kg, about 2 mg/kg to about 2.5mg/kg, about 2 mg/kg to about 10 mg/kg, about 3 mg/kg to about 5 mg/kg,about 3 mg/kg to about 10 mg/kg, about 3.5 mg/kg to about 5 mg/kg, about4 mg/kg to about 5 mg/kg, about 4.5 mg/kg to about 5 mg/kg, about 4mg/kg to about 10 mg/kg, about 4.5 mg/kg to about 10 mg/kg, about 5mg/kg to about 10 mg/kg, about 5.5 mg/kg to about 10 mg/kg, about 6mg/kg to about 10 mg/kg, about 6.5 mg/kg to about 10 mg/kg, about 7mg/kg to about 10 mg/kg, about 7.5 mg/kg to about 10 mg/kg, about 8mg/kg to about 10 mg/kg, about 8.5 mg/kg to about 10 mg/kg, about 9mg/kg to about 10 mg/kg, or about 9.5 mg/kg to about 10 mg/kg. Valuesand ranges intermediate to the recited values are also intended to bepart of this invention.

For example, the dsRNA may be administered at a dose of about 0.1, 0.2,0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7,1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2,3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7,4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2,6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7,7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2,9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or about 10 mg/kg. Values and rangesintermediate to the recited values are also intended to be part of thisinvention.

In other embodiments, for example, when a composition of the inventioncomprises a dsRNA as described herein and an N-acetylgalactosamine,subjects can be administered a therapeutic amount of iRNA, such as adose of about 0.1 to about 50 mg/kg, about 0.25 to about 50 mg/kg, about0.5 to about 50 mg/kg, about 0.75 to about 50 mg/kg, about 1 to about 50mg/kg, about 1.5 to about 50 mg/kg, about 2 to about 50 mg/kg, about 2.5to about 50 mg/kg, about 3 to about 50 mg/kg, about 3.5 to about 50mg/kg, about 4 to about 50 mg/kg, about 4.5 to about 50 mg/kg, about 5to about 50 mg/kg, about 7.5 to about 50 mg/kg, about 10 to about 50mg/kg, about 15 to about 50 mg/kg, about 20 to about 50 mg/kg, about 20to about 50 mg/kg, about 25 to about 50 mg/kg, about 25 to about 50mg/kg, about 30 to about 50 mg/kg, about 35 to about 50 mg/kg, about 40to about 50 mg/kg, about 45 to about 50 mg/kg, about 0.1 to about 45mg/kg, about 0.25 to about 45 mg/kg, about 0.5 to about 45 mg/kg, about0.75 to about 45 mg/kg, about 1 to about 45 mg/kg, about 1.5 to about 45mg/kg, about 2 to about 45 mg/kg, about 2.5 to about 45 mg/kg, about 3to about 45 mg/kg, about 3.5 to about 45 mg/kg, about 4 to about 45mg/kg, about 4.5 to about 45 mg/kg, about 5 to about 45 mg/kg, about 7.5to about 45 mg/kg, about 10 to about 45 mg/kg, about 15 to about 45mg/kg, about 20 to about 45 mg/kg, about 20 to about 45 mg/kg, about 25to about 45 mg/kg, about 25 to about 45 mg/kg, about 30 to about 45mg/kg, about 35 to about 45 mg/kg, about 40 to about 45 mg/kg, about 0.1to about 40 mg/kg, about 0.25 to about 40 mg/kg, about 0.5 to about 40mg/kg, about 0.75 to about 40 mg/kg, about 1 to about 40 mg/kg, about1.5 to about 40 mg/kg, about 2 to about 40 mg/kg, about 2.5 to about 40mg/kg, about 3 to about 40 mg/kg, about 3.5 to about 40 mg/kg, about 4to about 40 mg/kg, about 4.5 to about 40 mg/kg, about 5 to about 40mg/kg, about 7.5 to about 40 mg/kg, about 10 to about 40 mg/kg, about 15to about 40 mg/kg, about 20 to about 40 mg/kg, about 20 to about 40mg/kg, about 25 to about 40 mg/kg, about 25 to about 40 mg/kg, about 30to about 40 mg/kg, about 35 to about 40 mg/kg, about 0.1 to about 30mg/kg, about 0.25 to about 30 mg/kg, about 0.5 to about 30 mg/kg, about0.75 to about 30 mg/kg, about 1 to about 30 mg/kg, about 1.5 to about 30mg/kg, about 2 to about 30 mg/kg, about 2.5 to about 30 mg/kg, about 3to about 30 mg/kg, about 3.5 to about 30 mg/kg, about 4 to about 30mg/kg, about 4.5 to about 30 mg/kg, about 5 to about 30 mg/kg, about 7.5to about 30 mg/kg, about 10 to about 30 mg/kg, about 15 to about 30mg/kg, about 20 to about 30 mg/kg, about 20 to about 30 mg/kg, about 25to about 30 mg/kg, about 0.1 to about 20 mg/kg, about 0.25 to about 20mg/kg, about 0.5 to about 20 mg/kg, about 0.75 to about 20 mg/kg, about1 to about 20 mg/kg, about 1.5 to about 20 mg/kg, about 2 to about 20mg/kg, about 2.5 to about 20 mg/kg, about 3 to about 20 mg/kg, about 3.5to about 20 mg/kg, about 4 to about 20 mg/kg, about 4.5 to about 20mg/kg, about 5 to about 20 mg/kg, about 7.5 to about 20 mg/kg, about 10to about 20 mg/kg, or about 15 to about 20 mg/kg. In one embodiment,when a composition of the invention comprises a dsRNA as describedherein and an N-acetylgalactosamine, subjects can be administered atherapeutic amount of about 10 to about 30 mg/kg of dsRNA. Values andranges intermediate to the recited values are also intended to be partof this invention.

For example, subjects can be administered a therapeutic amount of iRNA,such as about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2,1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7,2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2,4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7,5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2,7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7,8.8, 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10, 10.5, 11,11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18,18.5, 19, 19.5, 20, 20.5, 21, 21.5, 22, 22.5, 23, 23.5, 24, 24.5, 25,25.5, 26, 26.5, 27, 27.5, 28, 28.5, 29, 29.5, 30, 31, 32, 33, 34, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or about 50mg/kg. Values and ranges intermediate to the recited values are alsointended to be part of this invention.

In certain embodiments of the invention, for example, when adouble-stranded RNAi agent includes a modification (e.g., one or moremotifs of three identical modifications on three consecutivenucleotides), including one such motif at or near the cleavage site ofthe agent, six phosphorothioate linkages, and a ligand, such an agent isadministered at a dose of about 0.01 to about 0.5 mg/kg, about 0.01 toabout 0.4 mg/kg, about 0.01 to about 0.3 mg/kg, about 0.01 to about 0.2mg/kg, about 0.01 to about 0.1 mg/kg, about 0.01 mg/kg to about 0.09mg/kg, about 0.01 mg/kg to about 0.08 mg/kg, about 0.01 mg/kg to about0.07 mg/kg, about 0.01 mg/kg to about 0.06 mg/kg, about 0.01 mg/kg toabout 0.05 mg/kg, about 0.02 to about 0.5 mg/kg, about 0.02 to about 0.4mg/kg, about 0.02 to about 0.3 mg/kg, about 0.02 to about 0.2 mg/kg,about 0.02 to about 0.1 mg/kg, about 0.02 mg/kg to about 0.09 mg/kg,about 0.02 mg/kg to about 0.08 mg/kg, about 0.02 mg/kg to about 0.07mg/kg, about 0.02 mg/kg to about 0.06 mg/kg, about 0.02 mg/kg to about0.05 mg/kg, about 0.03 to about 0.5 mg/kg, about 0.03 to about 0.4mg/kg, about 0.03 to about 0.3 mg/kg, about 0.03 to about 0.2 mg/kg,about 0.03 to about 0.1 mg/kg, about 0.03 mg/kg to about 0.09 mg/kg,about 0.03 mg/kg to about 0.08 mg/kg, about 0.03 mg/kg to about 0.07mg/kg, about 0.03 mg/kg to about 0.06 mg/kg, about 0.03 mg/kg to about0.05 mg/kg, about 0.04 to about 0.5 mg/kg, about 0.04 to about 0.4mg/kg, about 0.04 to about 0.3 mg/kg, about 0.04 to about 0.2 mg/kg,about 0.04 to about 0.1 mg/kg, about 0.04 mg/kg to about 0.09 mg/kg,about 0.04 mg/kg to about 0.08 mg/kg, about 0.04 mg/kg to about 0.07mg/kg, about 0.04 mg/kg to about 0.06 mg/kg, about 0.05 to about 0.5mg/kg, about 0.05 to about 0.4 mg/kg, about 0.05 to about 0.3 mg/kg,about 0.05 to about 0.2 mg/kg, about 0.05 to about 0.1 mg/kg, about 0.05mg/kg to about 0.09 mg/kg, about 0.05 mg/kg to about 0.08 mg/kg, orabout 0.05 mg/kg to about 0.07 mg/kg. Values and ranges intermediate tothe foregoing recited values are also intended to be part of thisinvention, e.g., the RNAi agent may be administered to the subject at adose of about 0.015 mg/kg to about 0.45 mg/kg.

For example, the RNAi agent, e.g., RNAi agent in a pharmaceuticalcomposition, may be administered at a dose of about 0.01 mg/kg, 0.0125mg/kg, 0.015 mg/kg, 0.0175 mg/kg, 0.02 mg/kg, 0.0225 mg/kg, 0.025 mg/kg,0.0275 mg/kg, 0.03 mg/kg, 0.0325 mg/kg, 0.035 mg/kg, 0.0375 mg/kg, 0.04mg/kg, 0.0425 mg/kg, 0.045 mg/kg, 0.0475 mg/kg, 0.05 mg/kg, 0.0525mg/kg, 0.055 mg/kg, 0.0575 mg/kg, 0.06 mg/kg, 0.0625 mg/kg, 0.065 mg/kg,0.0675 mg/kg, 0.07 mg/kg, 0.0725 mg/kg, 0.075 mg/kg, 0.0775 mg/kg, 0.08mg/kg, 0.0825 mg/kg, 0.085 mg/kg, 0.0875 mg/kg, 0.09 mg/kg, 0.0925mg/kg, 0.095 mg/kg, 0.0975 mg/kg, 0.1 mg/kg, 0.125 mg/kg, 0.15 mg/kg,0.175 mg/kg, 0.2 mg/kg, 0.225 mg/kg, 0.25 mg/kg, 0.275 mg/kg, 0.3 mg/kg,0.325 mg/kg, 0.35 mg/kg, 0.375 mg/kg, 0.4 mg/kg, 0.425 mg/kg, 0.45mg/kg, 0.475 mg/kg, or about 0.5 mg/kg. Values intermediate to theforegoing recited values are also intended to be part of this invention.

In some embodiments, the RNAi agent is administered as a fixed dose ofbetween about 100 mg to about 900 mg, e.g., between about 100 mg toabout 850 mg, between about 100 mg to about 800 mg, between about 100 mgto about 750 mg, between about 100 mg to about 700 mg, between about 100mg to about 650 mg, between about 100 mg to about 600 mg, between about100 mg to about 550 mg, between about 100 mg to about 500 mg, betweenabout 200 mg to about 850 mg, between about 200 mg to about 800 mg,between about 200 mg to about 750 mg, between about 200 mg to about 700mg, between about 200 mg to about 650 mg, between about 200 mg to about600 mg, between about 200 mg to about 550 mg, between about 200 mg toabout 500 mg, between about 300 mg to about 850 mg, between about 300 mgto about 800 mg, between about 300 mg to about 750 mg, between about 300mg to about 700 mg, between about 300 mg to about 650 mg, between about300 mg to about 600 mg, between about 300 mg to about 550 mg, betweenabout 300 mg to about 500 mg, between about 400 mg to about 850 mg,between about 400 mg to about 800 mg, between about 400 mg to about 750mg, between about 400 mg to about 700 mg, between about 400 mg to about650 mg, between about 400 mg to about 600 mg, between about 400 mg toabout 550 mg, or between about 400 mg to about 500 mg.

In some embodiments, the RNAi agent is administered as a fixed dose ofabout 100 mg, about 125 mg, about 150 mg, about 175 mg, 200 mg, about225 mg, about 250 mg, about 275 mg, about 300 mg, about 325 mg, about350 mg, about 375 mg, about 400 mg, about 425 mg, about 450 mg, about475 mg, about 500 mg, about 525 mg, about 550 mg, about 575 mg, about600 mg, about 625 mg, about 650 mg, about 675 mg, about 700 mg, about725 mg, about 750 mg, about 775 mg, about 800 mg, about 825 mg, about850 mg, about 875 mg, or about 900 mg.

The iRNA can be administered by intravenous infusion over a period oftime, such as over a 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, or about a 25 minute period. The administrationmay be repeated, for example, on a regular basis, such as weekly,biweekly (i.e., every two weeks) for one month, two months, threemonths, four months or longer. After an initial treatment regimen, thetreatments can be administered on a less frequent basis. For example,after administration weekly or biweekly for three months, administrationcan be repeated once per month, for six months or a year or longer.

Administration of the iRNA can reduce the presence of serum and/or liverHDV DNA, the presence of serum and/or liver HDV antigen e.g., in a cell,tissue, blood, urine or other compartment of the patient by at leastabout 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%,19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%,33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%,47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%,61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 72%, 73%,75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least about 99%or more, e.g., to below the level of detection of the assay.

Before administration of a full dose of the iRNA, patients can beadministered a smaller dose, such as a 5% infusion, and monitored foradverse effects, such as an allergic reaction. In another example, thepatient can be monitored for unwanted immunostimulatory effects, such asincreased cytokine (e.g., TNF-alpha or INF-alpha) levels.

The methods of treatment of HDV can further comprise treatment of HBVusing any method known in the art. In certain embodiments, methods oftreatment of HBV comprise administration of an iRNA compound, e.g., aniRNA agent targeting an HBV gene as provided herein.

Owing to the inhibitory effects on HDV expression, a compositionaccording to the invention or a pharmaceutical composition preparedtherefrom can enhance the quality of life.

An iRNA of the invention may be administered in “naked” form, where themodified or unmodified iRNA agent is directly suspended in aqueous orsuitable buffer solvent, as a “free iRNA.” A free iRNA is administeredin the absence of a pharmaceutical composition. The free iRNA may be ina suitable buffer solution. The buffer solution may comprise acetate,citrate, prolamine, carbonate, or phosphate, or any combination thereof.In one embodiment, the buffer solution is phosphate buffered saline(PBS). The pH and osmolarity of the buffer solution containing the iRNAcan be adjusted such that it is suitable for administering to a subject.

Alternatively, an iRNA of the invention may be administered as apharmaceutical composition, such as a dsRNA liposomal formulation.

Subjects that would benefit from a reduction and/or inhibition of HDVgene expression are those having an HDV infection and/or anHDV-associated disease or disorder as described herein.

Treatment of a subject that would benefit from a reduction and/orinhibition of HDV gene expression includes therapeutic and prophylactictreatment.

The invention further provides methods and uses of an iRNA agent or apharmaceutical composition thereof for treating a subject that wouldbenefit from reduction and/or inhibition of HDV gene expression, e.g., asubject having a HDV-associated disease, in combination with otherpharmaceuticals and/or other therapeutic methods, e.g., with knownpharmaceuticals and/or known therapeutic methods, such as, for example,those which are currently employed for treating these disorders.

For example, a subject administered a first RNAi agent or a first andsecond RNAi agent targeting HDV may further be administered one or moreiRNA agents targeting HBV and/or one or more agents which function by anon-iRNA mechanism and which are useful in treating an HBV and/or an HDVinfection. Exemplary agents include immune modulators which stimulatethe immune system by, for example, enhancing T-cell helper activity,maturation of B lymphocytes, inhibiting T-cell suppressors, andenhancing HLA type I expression. Suitable immune modulators includeinterferons which have a variety of properties that include antiviral,immunomodulatory, and antiproliferative effects.

For example, the current treatment for chronic hepatitis B is interferontherapy. Interferon therapy is administered to subjects who have adocumented HBV infection for at least six months, elevated liver enzymes(AST and ALT) and an actively dividing virus in their blood (HBeAg,and/or HBV DNA positive tests). Interferon-α therapy produces along-term, sustained remission of the disease in about 35% of those withchronic hepatitis B, with normalization of liver enzymes and loss of thethree markers for an active infection (HBeAg, HBV DNA, and HBsAg).Subjects with an acute HBV infection, end stage cirrhosis or other majormedical problems are typically not treated with interferon.

In addition, interferon therapy for patients with HBV-related cirrhosisdecreases significantly the hepatocellular carcinoma (HCC) rate,particularly in patients with a larger amount of serum HBV DNA. Inpatients with HBeAg-positive compensated cirrhosis, virological andbiochemical remission following interferon therapy is associated withimproved survival. In patients with chronic HBV infection, the clearanceof HBeAg after treatment with interferon-α is associated with improvedclinical outcomes.

The standard duration of therapy is considered 16 weeks. Patients whoexhibit a low level of viral replication at the end of the standardregimen benefit most from prolonged treatment.

Thus, in some embodiments, an iRNA targeting one or more HDV genes isadministered in combination with, e.g., an agent useful in treating anHDV-associated disease as described elsewhere herein.

For example, additional therapeutics and therapeutic methods suitablefor treating a subject that would benefit from reduction in HDVexpression, e.g., a subject having a HDV-associated disease, include aniRNA agent targeting a different portion of the HDV genome, an antiviralagent, a nucleotide analog, a nucleoside analog, a reverse transcriptaseinhibitor (e.g., Tenofovir disoproxil fumarate (TDF), Tenofoviralafenamide, Lamivudine, Adefovir dipivoxil, Entecavir (ETV),Telbivudine, AGX-1009, emtricitabine, clevudine, ritonavir, dipivoxil,lobucavir, famvir, FTC, N-Acetyl-Cysteine (NAC), PC1323, theradigm-HBV,thymosin-alpha, and ganciclovir), an immune stimulator (e.g., pegylatedinterferon alfa 2a (PEG-IFN-α2a), Interferon alfa-2b, a recombinanthuman interleukin-7, and aToll-like receptor 7 (TLR7) agonist), atherapeutic vaccine (e.g., GS-4774, DV-601, and TG1050), a viral entryinhibitor (e.g., Myrcludex), an oligonucleotide that inhibits thesecretion or release of HbsAg (e.g., REP 9AC), a capsid inhibitor (e.g.,Bay41-4109 and NVR-1221), a cccDNA inhibitor (e.g., IHVR-25), or othertherapeutic agents and/or procedures, e.g., liver transplant,chemotherapy, for treating a HBV-associated disease, a combination ofany of the foregoing.

In certain embodiments, a first iRNA agent targeting one or more HDVgenes is administered in combination with a second iRNA agent targetinga different portion of the HDV genome. For example, the first RNAi agentcomprises a first sense strand and a first antisense strand forming adouble-stranded region, wherein substantially all of the nucleotides ofsaid first sense strand and substantially all of the nucleotides of thefirst antisense strand are modified nucleotides, wherein said firstsense strand is conjugated to a ligand attached at the 3′-terminus, andwherein the ligand is one or more GalNAc derivatives attached through abivalent or trivalent branched linker; and the second RNAi agentcomprises a second sense strand and a second antisense strand forming adouble-stranded region, wherein substantially all of the nucleotides ofthe second sense strand and substantially all of the nucleotides of thesecond antisense strand are modified nucleotides, wherein the secondsense strand is conjugated to a ligand attached at the 3′-terminus, andwherein the ligand is one or more GalNAc derivatives attached through abivalent or trivalent branched linker; wherein the first sense strandcomprises a sequences selected from the group consisting of any one ofthe sense sequences in any one of Tables 11, 12, 31, and 32 (or anucleotide sequence which is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, or 99% identical over its entire length to the foregoingnucleotide sequences), and wherein the first antisense strand comprisesa sequence selected from the group consisting of any one of theantisense sequences in any one of Tables 11, 12, 31, and 32 (or anucleotide sequence which is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, or 99% identical over its entire length to the foregoingnucleotide sequences); wherein the second sense strand comprises any oneof the sense sequences in any one of Tables 11, 12, 31, and 32 (or anucleotide sequence which is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, or 99% identical over its entire length to the foregoingnucleotide sequence), and wherein the second antisense strand comprisesany one of the antisense sequences in any one of Tables 11, 12, 31, and32 (or a nucleotide sequence which is at least 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, or 99% identical over its entire length to theforegoing nucleotide sequence).

In one embodiment, the first and second sense strands each independentlycomprise a sequence selected from the group consisting of any one of thesense sequences from AD-70260.1, AD-70232.1, AD-70249.1, AD-70244.1,AD-70272.1, AD-70228.1, AD-70255.1, AD-70278.1, AD-70295.1, AD-67200.1,AD-67211.1, AD-67199.1, AD-67202.1, AD-67208.1, AD-67210.1, AD-70259.1,AD-70267.1, AD-70272.1, AD-70271.1, AD-70268.1, AD-70269.1, AD-70232.1,AD-70256.1, AD-70257.1, or AD-70275.1 (or a nucleotide sequence which isat least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identicalover its entire length to the foregoing nucleotide sequences).

In another embodiment, the first and second antisense strands eachindependently comprise a sequence selected from the group consisting ofany one of the antisense sequences from AD-70260.1, AD-70232.1,AD-70249.1, AD-70244.1, AD-70272.1, AD-70228.1, AD-70255.1, AD-70278.1,AD-70295.1, AD-67200.1, AD-67211.1, AD-67199.1, AD-67202.1, AD-67208.1,AD-67210.1, AD-70259.1, AD-70267.1, AD-70272.1, AD-70271.1, AD-70268.1,AD-70269.1, AD-70232.1, AD-70256.1, AD-70257.1, or AD-70275.1 (or anucleotide sequence which is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, or 99% identical over its entire length to the foregoingnucleotide sequences).

In one embodiment, all of the nucleotides of the first and second sensestrand and/or all of the nucleotides of the first and second antisensestrand comprise a modification.

In one embodiment, the at least one of the modified nucleotides isselected from the group consisting of a 3′-terminal deoxy-thymine (dT)nucleotide, a 2′-O-methyl modified nucleotide, a 2′-fluoro modifiednucleotide, a 2′-deoxy-modified nucleotide, a locked nucleotide, anunlocked nucleotide, a conformationally restricted nucleotide, aconstrained ethyl nucleotide, an abasic nucleotide, a 2′-amino-modifiednucleotide, a 2′-O-allyl-modified nucleotide, 2′-C-alkyl-modifiednucleotide, 2′-hydroxyl-modified nucleotide, a 2′-methoxyethyl modifiednucleotide, a 2′-O-alkyl-modified nucleotide, a morpholino nucleotide, aphosphoramidate, a non-natural base comprising nucleotide, atetrahydropyran modified nucleotide, a 1,5-anhydrohexitol modifiednucleotide, a cyclohexenyl modified nucleotide, a nucleotide comprisinga phosphorothioate group, a nucleotide comprising a methylphosphonategroup, a nucleotide comprising a 5′-phosphate, and a nucleotidecomprising a 5′-phosphate mimic.

A subject administered a first or a first and second RNAi agent mayfurther be administered one or more iRNA agents targeting HBV antiviralagent, a reverse transcriptase inhibitor (e.g., Tenofovir disoproxilfumarate (TDF), Tenofovir alafenamide, Lamivudine, Adefovir dipivoxil,Entecavir (ETV), Telbivudine, and AGX-1009), an immune stimulator (e.g.,pegylated interferon alfa 2a (PEG-IFN-α2a), Interferon alfa-2b, arecombinant human interleukin-7, and aToll-like receptor 7 (TLR7)agonist), a therapeutic vaccine (e.g., GS-4774, DV-601, and TG1050), aviral entry inhibitor (e.g., Myrcludex), an oligonucleotide thatinhibits the secretion or release of HbsAg (e.g., REP 9AC), a capsidinhibitor (e.g., Bay41-4109 and NVR-1221), a cccDNA inhibitor (e.g.,IHVR-25), or other therapeutic agents and/or procedures, e.g., livertransplant, chemotherapy, for treating a HDV-associated disease, acombination of any of the foregoing.

In one embodiment, the methods of the invention include administering toa subject having an HDV infection and/or HDV-associated disease areverse transcriptase inhibitor. In another embodiment, the methods ofthe invention include administering to a subject having an HDV infectionand/or HDV-associate disease a reverse transcriptase inhibitor and animmune stimulator.

The iRNA agent and an additional therapeutic agent and/or treatment maybe administered at the same time and/or in the same combination, e.g.,parenterally, or the additional therapeutic agent can be administered aspart of a separate composition or at separate times and/or by anothermethod known in the art or described herein.

The present invention also provides methods of using an iRNA agent ofthe invention and/or a composition containing an iRNA agent of theinvention to reduce and/or inhibit HDV expression in a cell. In otheraspects, the present invention provides an iRNA of the invention and/ora composition comprising an iRNA of the invention for use in reducingand/or inhibiting HDV gene expression in a cell. In yet other aspects,use of an iRNA of the invention and/or a composition comprising an iRNAof the invention for the manufacture of a medicament for reducing and/orinhibiting HDV gene expression in a cell are provided. In still otheraspect, the the present invention provides an iRNA of the inventionand/or a composition comprising an iRNA of the invention for use inreducing and/or inhibiting HDV replication in a cell. In yet otheraspects, use of an iRNA of the invention and/or a composition comprisingan iRNA of the invention for the manufacture of a medicament forreducing and/or inhibiting HDV replication in a cell are provided. Themethods and uses include contacting the cell with an iRNA, e.g., adsRNA, of the invention and maintaining the cell for a time sufficientto obtain degradation of the mRNA transcript of an HDV gene, therebyinhibiting expression of the HDV gene or inhibiting HDV replication inthe cell.

Reduction in gene expression can be assessed by any methods known in theart. For example, a reduction in the expression of HDV may be determinedby determining the mRNA expression level of HDV using methods routine toone of ordinary skill in the art, e.g., Northern blotting, qRT-PCR, bydetermining the protein level of HDV using methods routine to one ofordinary skill in the art, such as western blotting, immunologicaltechniques, flow cytometry methods, ELISA, and/or by determining abiological activity of HDV.

In the methods and uses of the invention the cell may be contacted invitro or in vivo, i.e., the cell may be within a subject.

A cell suitable for treatment using the methods of the invention may beany cell that expresses an HDV gene, e.g., a cell infected with HDV or acell comprising an expression vector comprising an HDV genome or portionof an HDV gene. A cell suitable for use in the methods and uses of theinvention may be a mammalian cell, e.g., a primate cell (such as a humancell or a non-human primate cell, e.g., a monkey cell or a chimpanzeecell), a non-primate cell (such as a cow cell, a pig cell, a camel cell,a llama cell, a horse cell, a goat cell, a rabbit cell, a sheep cell, ahamster, a guinea pig cell, a cat cell, a dog cell, a rat cell, a mousecell, a lion cell, a tiger cell, a bear cell, or a buffalo cell), a birdcell (e.g., a duck cell or a goose cell), or a whale cell. In oneembodiment, the cell is a human cell, e.g., a human liver cell.

HDV gene expression may be inhibited in the cell by at least about 5%,6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%,21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%,35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%,49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%,63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%,77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or about 100%, i.e., tobelow the level of detection of the assay.

HDV replication may be inhibited in the cell by at least about 5%, 6%,7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%,22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%,36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%,50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%,64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 72%, 74%, 75%, 76%, 77%,78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or about 100%, i.e., to belowthe level of detection of the assay.

The in vivo methods and uses of the invention may include administeringto a subject a composition containing an iRNA, where the iRNA includes anucleotide sequence that is complementary to at least a part of an RNAtranscript of the HDV gene of the mammal to be treated. When theorganism to be treated is a human, the composition can be administeredby any means known in the art including, but not limited tosubcutaneous, intravenous, oral, intraperitoneal, or parenteral routes,including intracranial (e.g., intraventricular, intraparenchymal andintrathecal), intramuscular, transdermal, airway (aerosol), nasal,rectal, and topical (including buccal and sublingual) administration. Incertain embodiments, the compositions are administered by subcutaneousinjection. In some embodiments, the compositions are administered byintravenous infusion or injection. In other embodiments, thecompositions are administered by intramuscular injection.

In some embodiments, the administration is via a depot injection. Adepot injection may release the iRNA in a consistent way over aprolonged time period. Thus, a depot injection may reduce the frequencyof dosing needed to obtain a desired effect, e.g., a desired inhibitionof HDV, or a therapeutic or prophylactic effect. A depot injection mayalso provide more consistent serum concentrations. Depot injections mayinclude subcutaneous injections or intramuscular injections. Inpreferred embodiments, the depot injection is a subcutaneous injection.

In some embodiments, the administration is via a pump. The pump may bean external pump or a surgically implanted pump. In certain embodiments,the pump is a subcutaneously implanted osmotic pump. In otherembodiments, the pump is an infusion pump. An infusion pump may be usedfor intravenous, subcutaneous, arterial, or epidural infusions. Inpreferred embodiments, the infusion pump is a subcutaneous infusionpump. In other embodiments, the pump is a surgically implanted pump thatdelivers the iRNA to the liver.

The mode of administration may be chosen based upon whether local orsystemic treatment is desired and based upon the area to be treated. Theroute and site of administration may be chosen to enhance targeting.

In one aspect, the present invention also provides methods forinhibiting the expression of an HDV gene in a mammal, e.g., a human. Thepresent invention also provides a composition comprising an iRNA, e.g.,a dsRNA, that targets an HDV gene in a cell of a mammal for use ininhibiting expression of the HDV gene in the mammal. In another aspect,the present invention provides use of an iRNA, e.g., a dsRNA, thattargets an HDV gene in a cell of a mammal in the manufacture of amedicament for inhibiting expression of the HDV gene in the mammal.

The methods and uses include administering to the mammal, e.g., a human,a composition comprising an iRNA, e.g., a dsRNA, that targets an HDVgene in a cell of the mammal and maintaining the mammal for a timesufficient to obtain degradation of the mRNA transcript of the HDV gene,thereby inhibiting expression of the HDV gene in the mammal.

Reduction in gene expression can be assessed in peripheral blood sampleof the iRNA-administered subject by any methods known it the art, e.g.qRT-PCR, described herein. Reduction in protein production can beassessed by any methods known it the art and by methods, e.g., ELISA orwestern blotting, described herein. In one embodiment, a puncture liverbiopsy sample serves as the tissue material for monitoring the reductionin HDV gene and/or protein expression. In another embodiment, a bloodsample serves as the tissue material for monitoring the reduction in HDVgene and/or protein expression.

In one embodiment, verification of RISC medicated cleavage of target invivo following administration of iRNA agent is done by performing5′-RACE or modifications of the protocol as known in the art (Lasham Aet al., (2010) Nucleic Acid Res., 38 (3) p-e19) (Zimmermann et al.(2006) Nature 441: 111-4).

This invention is further illustrated by the following examples whichshould not be construed as limiting. The entire contents of allreferences, patents and published patent applications cited throughoutthis application, as well as the Figures and the Sequence Listing, arehereby incorporated herein by reference.

EXAMPLES Example 1. iRNA Synthesis Source of Reagents

Where the source of a reagent is not specifically given herein, suchreagent can be obtained from any supplier of reagents for molecularbiology at a quality/purity standard for application in molecularbiology.

Transcripts

siRNA Design

The selection of siRNA designs targeting HBV was driven by two primaryfactors: a) potency and b), the desire to employ siRNA with near-perfectmatches with greater than 90% fractional coverage of the large number ofpublic HBV sequences of all known serotypes (A through H). Thecoordinates for the siRNA selection were determined relative to the NCBIHBV reference genome sequence NC_003977.1 (GenBank Accession No.GI:21326584 (SEQ ID NO:1). The reverse complement of SEQ ID NO:1 isprovided in SEQ ID NO:2. A first set of siRNAs containingstructure-activity modifications, including various 2′-O-methyl and2′-fluoro substitution patterns, centered on two adjacent regions of theHBV genome coding for surface antigen (HbSAg) and the HBV polymerase,were designed, synthesized and screened in-vitro. A second set of siRNAswere designed, synthesized and screened targeting additional targetregions with particular attention to positions 1581-1599 of SEQ ID NO:1that code, in addition to the HbSAg and polymerase, the X gene.

The sequence of Hepatitis B virus genomic DNA, complete sequence,isolate 22Y04HCC (GenBank Accession No., GI: 3582357) and its reversecomplement are provided in SEQ ID NOs. 3 and 4, respectively.

A detailed list of the unmodified HBV sense and antisense strandsequences is shown in Tables 3, 14, 24, and 27.

A detailed list of the modified HBV sense and antisense strand sequencesis shown in Tables 4, 6, 15, 25, and 28.

siRNA Synthesis

HBV siRNA sequences were synthesized at 1 μmol scale on Mermade 192synthesizer (BioAutomation) using the solid support mediatedphosphoramidite chemistry. The solid support was controlled pore glass(500 A) loaded with custom GalNAc ligand or universal solid support (AMbiochemical). Ancillary synthesis reagents, 2′-F and 2′-O-Methyl RNA anddeoxy phosphoramidites were obtained from Thermo-Fisher (Milwaukee,Wis.) and Hongene (China). 2′F 2′-O-Methyl, GNA (glycol nucleic acids),5′phosphate and abasic modifications were introduced employing thecorresponding phosphoramidites. Synthesis of 3′ GalNAc conjugated singlestrands was performed on a GalNAc modified CPG support. Custom CPGuniversal solid support was used for the synthesis of antisense singlestrands. Coupling time for all phosphoramidites (100 mM in acetonitrile)was 5 min employing 5-Ethylthio-1H-tetrazole (ETT) as activator (0.6 Min acetonitrile). Phosphorothioate linkages were generated using a 50 mMsolution of 3-((Dimethylamino-methylidene)amino)-3H-1,2,4-dithiazole-3-thione (DDTT, obtained from Chemgenes(Wilmington, Mass., USA)) in anhydrous acetonitrile/pyridine (1:1 v/v).Oxidation time was 3 minutes. All sequences were synthesized with finalremoval of the DMT group (“DMT off”).

Upon completion of the solid phase synthesis, oligoribonucleotides werecleaved from the solid support and deprotected in sealed 96 deep wellplates using 200 μL Aqueous Methylamine reagents at 60° C. for 20minutes. At the end of cleavage and deprotection step, the synthesisplate was allowed to come to room temperature and was precipitated byaddition of 1 mL of acetontile: ethanol mixture (9:1). The plates werecooled at −80 C for 2 hrs, supernatant decanted carefully with the aidof a multi channel pipette. The oligonucleotide pellet was re-suspendedin 20 mM NaOAc buffer and were desalted using a 5 mL HiTrap sizeexclusion column (GE Healthcare) on an AKTA Purifier System equippedwith an A905 autosampler and a Frac 950 fraction collector. Desaltedsamples were collected in 96-well plates. Samples from each sequencewere analyzed by LC-MS to confirm the identity, UV (260 nm) forquantification and a selected set of samples by IEX chromatography todetermine purity.

Annealing of HBV single strands was performed on a Tecan liquid handlingrobot. Equimolar mixture of sense and antisense single strands werecombined and annealed in 96 well plates. After combining thecomplementary single strands, the 96-well plate was sealed tightly andheated in an oven at 100° C. for 10 minutes and allowed to come slowlyto room temperature over a period 2-3 hours. The concentration of eachduplex was normalized to 10M in 1×PBS.

Example 2. In Vitro Screening of siRNA Duplexes Cell Culture andTransfections

Cos7 cells (ATCC, Manassas, Va.) were grown to near confluence at 37° C.in an atmosphere of 5% CO2 in DMEM (ATCC) supplemented with 10% FBS,before being released from the plate by trypsinization. Dual-Glo®Luciferaseconstructs generated in the psiCHECK2 plasmid containingapproximately 1.1 kb of HBV genomic sequences were transfected intoapproximately 15×10⁴ cells using Lipofectamine 2000 (Invitrogen,Carlsbad Calif. cat #11668-019). For each well of a 96 well plate, 0.2μl of Lipofectamine was added to 10 ng of plasmid vector in 14.8 μl ofOpti-MEM and allowed to complex at room temperature for 15 minutes. Themixture was then added to the cells which were resuspended in 80 μl offresh complete media. After approximately 24 hours, the media wereremoved and the cells re-transfected with siRNA. Each siRNA wastransfected into cells that had previously been transfected with thepsiCHECK2-HBV vector that had a perfect match for the siRNA. siRNAtransfection was carried out by adding 14.8 μl of Opti-MEM plus 0.2 μlof Lipofectamine RNAiMax per well (Invitrogen, Carlsbad Calif. cat#13778-150) to 5 μl of siRNA duplexes per well into a 96-well plate andincubated at room temperature for 15 minutes. The mixture was then addedto the cells previously transfected with the psiCHECK2-HBV plasmid thathad a perfect match to the siRNA sequence. Cells were incubated for 24hours before luciferase was measured.

Single dose experiments were performed at 10 nM and 0.01 nM final duplexconcentration.

Dual-Glo® Luciferase Assay

Twenty-four hours after the siRNAs were transfected, Firefly(transfection control) and Rinella (fused to HBV target sequence)luciferase were measured. First, media was removed from cells. ThenFirefly luciferase activity was measured by adding 75 μl of Dual-Glo®Luciferase Reagent equal to the culture medium volume to each well andmix. The mixture was incubated at room temperature for 30 minutes beforelunimescense (500 nm) was measured on a Spectramax (Molecular Devices)to detect the Firefly luciferase signal. Renilla luciferase activity wasmeasured by adding 75 μl of room temperature of Dual-Glo® Stop & Glo®Reagent was added to each well and the plates were incubated for 10-15minutes before luminescence was again measured to determine the Renillaluciferase signal. The Dual-Glo® Stop & Glo® Reagent, quench the fireflyluciferase signal and sustain luminescence for the Renilla luciferasereaction. siRNA activity was determined by normalizing the Renilla (HBV)signal to the Firefly (control) signal within each well. The magnitudeof siRNA activity was then assessed relative to cells that weretransfected with the same vector but were not treated with siRNA or weretreated with a non-targeting siRNA. All transfections were done at n=2or greater.

Essentially the same methods were used to screen siRNA targeted to HDV.

Table 5 shows the results of a single dose screen in Cos7 cellstransfected with the indicated HBV iRNAs. Data are expressed as percentof mRNA remaining relative to negative control.

TABLE 2 Abbreviations of nucleotide monomers used in nucleic acidsequence representation. It will be understood that these monomers, whenpresent in an oligonucleotide, are mutually linked by5'-3'-phosphodiester bonds. Abbreviation Nucleotide(s) AAdenosine-3'-phosphate Af 2'-fluoroadenosine-3'-phosphate Afs2'-fluoroadenosine-3'-phosphorothioate As adenosine-3'-phosphorothioateC cytidine-3'-phosphate Cf 2'-fluorocytidine-3'-phosphate Cfs2'-fluorocytidine-3'-phosphorothioate Cs cytidine-3'-phosphorothioate Gguanosine-3'-phosphate Gf 2'-fluoroguanosine-3'-phosphate Gfs2'-fluoroguanosine-3'-phosphorothioate Gs guanosine-3'-phosphorothioateT 5'-methyluridine-3'-phosphate Tf2'-fluoro-5-methyluridine-3'-phosphate Tfs2'-fluoro-5-methyluridine-3'-phosphorothioate Ts5-methyluridine-3'-phosphorothioate U Uridine-3'-phosphate Uf2'-fluorouridine-3'-phosphate Ufs 2'-fluorouridine-3'-phosphorothioateUs uridine-3'-phosphorothioate N any nucleotide (G, A, C, T or U) a2'-O-methyladenosine-3'-phosphate as2'-O-methyladenosine-3'-phosphorothioate c2'-O-methylcytidine-3'-phosphate cs2'-O-methylcytidine-3'-phosphorothioate g2'-O-methylguanosine-3'-phosphate gs2'-O-methylguanosine-3'-phosphorothioate t2'-O-methyl-5-methyluridine-3'-phosphate ts2'-O-methyl-5-methyluridine-3'-phosphorothioate u2'-O-methyluridine-3'-phosphate us2'-O-methyluridine-3'-phosphorothioate s phosphorothioate linkage L96N-[tris(GalNAc-alkyl)-amidodecanoyl)]-4- hydroxyprolinolHyp-(GalNAc-alkyl)3 (dt) deoxy-thymine Y342-hydroxymethyl-tetrahydrofurane-4-methoxy- 3-phosphate (abasic 2'-OMefuranose) Y44 2-hydroxymethyl-tetrahydrofurane-5-phosphate (Agn)Adenosine-glycol nucleic acid (GNA) (Tgn) Thymidine-glycol nucleic acid(GNA) S-Isomer (Cgn) Cytidine-glycol nucleic acid (GNA) P Phosphate VPVinyl-phosphate

TABLE 3 Unmodified Sense and Antisense Strand Sequences of HBV dsRNAsSense SEQ SEQ Position Duplex Oligo Sense Sequence ID AntisenseAntisense Sequence ID in NC_ Name Name (5′ to 3′) NO: Oligo Name(5′ to 3′) NO: 003977.1 AD-61522.2 A-123463.2 AGUUAUAUGGAUGAUGUGGUA 121A-123464.2 UACCACAUCAUCCAUAUAACUGA 556 731_753 AD-61547.2 A-123487.2GGAUGUGUCUGCGGCGUUUUA 122 A-123488.2 UAAAACGCCGCAGACACAUCCAG 557 373_395AD-63938.2 A-127896.1 ACUCGUGGUGGACUUCUCUCA 123 A-127897.1UGAGAGAAGUCCACCACGAGUCU 558 250_272 AD-63939.2 A-127909.1ACUCGUGGUGGACUUCUCUCA 124 A-127906.3 UGAGAGAAGUCCACCACGAGUCU 559 250_272AD-63940.2 A-127917.1 ACUCGUGGUGGACUUCTCUCA 125 A-127906.11UGAGAGAAGUCCACCACGAGUCU 560 250_272 AD-63940.3 A-127917.4ACUCGUGGUGGACUUCTCUCA 126 A-127906.19 UGAGAGAAGUCCACCACGAGUCU 561250_272 AD-63941.2 A-127905.8 ACUCGUGGUGGACUUCUCUCA 127 A-127925.1UGAGAGAAGUCCACCACGAGUCU 562 250_272 AD-63942.2 A-127933.1UCGUGGUGGACUUCUCUCA 128 A-127934.1 UGAGAGAAGUCCACCACGAGU 563 252_274AD-63943.2 A-127944.2 ACUCGUGGUGGACUUCUCUCA 129 A-127942.2UGAGAGAAGUCCACCACGAGUCU 564 250_272 AD-63945.2 A-127910.1ACUCGUGGUGGACUUCUCUCA 130 A-127906.4 UGAGAGAAGUCCACCACGAGUCU 565 250_272AD-63946.2 A-127918.1 ACUCGUGGUGGACUUCUCUCA 131 A-127906.12UGAGAGAAGUCCACCACGAGUCU 566 250_272 AD-63947.2 A-127905.9ACUCGUGGUGGACUUCUCUCA 132 A-127926.1 UGAGAGAAGUCCACCACGAGUCU 567 250_272AD-63948.2 A-127935.1 GUGGUGGACUUCUCUCA 133 A-127936.1UGAGAGAAGUCCACCACGA 568 254_276 AD-63949.2 A-127944.3ACUCGUGGUGGACUUCUCUCA 134 A-127906.14 UGAGAGAAGUCCACCACGAGUCU 569250_272 AD-63950.2 A-127900.1 UCGUGGUGGACUUCUCUCAUU 135 A-127901.1UGAGAGAAGUCCACCACGAUU 570 252_274 AD-63951.2 A-127911.1ACUCGUGGUGGACUUCUCUCA 136 A-127906.5 UGAGAGAAGUCCACCACGAGUCU 571 250_272AD-63952.2 A-127905.2 ACUCGUGGUGGACUUCUCUCA 137 A-127919.1UGAGAGAAGUCCACCACGAGUCU 572 250_272 AD-63953.2 A-127905.10ACUCGUGGUGGACUUCUCUCA 138 A-127927.1 UGAGAGAAGUCCACCACGAGUCU 573 250_272AD-63955.2 A-127945.1 ACUCGUGGUGGACUUCUCUCA 139 A-127940.3UGAGAGAAGUCCACCACGAGUCU 574 250_272 AD-63956.2 A-127902.1UCGUGGUGGACUUCUCUCA 140 A-127903.1 UGAGAGAAGUCCACCACGAUU 575 252_274AD-63957.2 A-127912.1 ACUCGUGGUGGACUUCUCUCA 141 A-127906.6UGAGAGAAGUCCACCACGAGUCU 576 250_272 AD-63958.2 A-127905.3ACUCGUGGUGGACUUCUCUCA 142 A-127920.1 UGAGAGAAGUCCACCACGAGUCU 577 250_272AD-63959.2 A-127905.11 ACUCGUGGUGGACUUCUCUCA 143 A-127928.1UGAGAGAAGUCCACCACGAGUCU 578 250_272 AD-63960.2 A-126619.2UAUUUCCUAGGGUACAA 144 A-127938.1 UGAGAGAAGUCCACCACGA 579 254_276AD-63961.2 A-127945.2 ACUCGUGGUGGACUUCUCUCA 145 A-127942.3UGAGAGAAGUCCACCACGAGUCU 580 250_272 AD-63962.2 A-127902.2UCGUGGUGGACUUCUCUCA 146 A-127904.1 UGAGAGAAGUCCACCACGAUU 581 252_274AD-63963.2 A-127913.1 ACUCGUGGUGGACUUCUCUCA 147 A-127906.7UGAGAGAAGUCCACCACGAGUCU 582 250_272 AD-63964.2 A-127905.4ACUCGUGGUGGACUUCUCUCA 148 A-127921.1 UGAGAGAAGUCCACCACGAGUCU 583 250_272AD-63965.2 A-127905.12 ACUCGUGGUGGACUUCUCUCA 149 A-127929.1UGAGAGAAGUCCACCACGAGUCU 584 250_272 AD-63966.2 A-127939.1ACUCGUGGUGGACUUCUCUCA 150 A-127940.1 UGAGAGAAGUCCACCACGAGUCU 585 250_272AD-63967.2 A-127945.3 ACUCGUGGUGGACUUCUCUCA 151 A-127906.15UGAGAGAAGUCCACCACGAGUCU 586 250_272 AD-63968.2 A-127905.1ACUCGUGGUGGACUUCUCUCA 152 A-127906.1 UGAGAGAAGUCCACCACGAGUCU 587 250_272AD-63968.2 A-127905.1 ACUCGUGGUGGACUUCUCUCA 153 A-127906.1UGAGAGAAGUCCACCACGAGUCU 588 250_272 AD-63968.4 A-127905.15ACUCGUGGUGGACUUCUCUCA 154 A-127906.17 UGAGAGAAGUCCACCACGAGUCU 589250_272 AD-63968.5 A-127905.17 ACUCGUGGUGGACUUCUCUCA 155 A-127906.18UGAGAGAAGUCCACCACGAGUCU 590 250_272 AD-63969.2 A-127914.1ACUCGUGGUGGACUUCUCUCA 156 A-127906.8 UGAGAGAAGUCCACCACGAGUCU 591 250_272AD-63970.2 A-127905.5 ACUCGUGGUGGACUUCUCUCA 157 A-127922.1UGAGAGAAGUCCACCACGAGUCU 592 250_272 AD-63971.2 A-127905.13ACUCGUGGUGGACUUCUCUCA 158 A-127930.1 UGAGAGAAGUCCACCACGAGUCU 593 250_272AD-63972.2 A-127941.1 ACUCGUGGUGGACUUCUCUCA 159 A-127942.1UGAGAGAAGUCCACCACGAGUCU 594 250_272 AD-63973.2 A-127946.1ACUCGUGGUGGACUUCUCUCA 160 A-127947.1 UGAGAGAAGTCCACCACGAGUCU 595 250_272AD-63975.2 A-127915.1 ACUCGUGGUGGACUUCTCUCA 161 A-127906.9UGAGAGAAGUCCACCACGAGUCU 596 250_272 AD-63976.2 A-127905.6ACUCGUGGUGGACUUCUCUCA 162 A-127923.1 UGAGAGAAGUCCACCACGAGUCU 597 250_272AD-63977.2 A-127917.2 ACUCGUGGUGGACUUCTCUCA 163 A-127931.1UGAGAGAAGUCCACCACGAGUCU 598 250_272 AD-63978.2 A-127943.1ACUCGUGGUGGACUUCUCUCA 164 A-127906.13 UGAGAGAAGUCCACCACGAGUCU 599250_272 AD-63979.2 A-127908.1 ACUCGUGGUGGACUUCUCUCA 165 A-127906.2UGAGAGAAGUCCACCACGAGUCU 600 250_272 AD-63980.2 A-127916.1ACUCGUGGUGGACUUCTCUCA 166 A-127906.10 UGAGAGAAGUCCACCACGAGUCU 601250_272 AD-63981.2 A-127905.7 ACUCGUGGUGGACUUCUCUCA 167 A-127924.1UGAGAGAAGUCCACCACGAGUCU 602 250_272 AD-63982.2 A-127917.3ACUCGUGGUGGACUUCTCUCA 168 A-127932.1 UGAGAGAAGUCCACCACGAGUCU 603 250_272AD-63983.2 A-127944.1 ACUCGUGGUGGACUUCUCUCA 169 A-127940.2UGAGAGAAGUCCACCACGAGUCU 604 250_272 AD-63985.2 A-127961.1GUGGUGGACUUCUCUCAAUUU 170 A-127956.4 AAAUUGAGAGAAGUCCACCACGA 605 254_276AD-63986.2 A-127969.1 GUGGUGGACUUCUCUCAAUUU 171 A-127956.12AAAUUGAGAGAAGUCCACCACGA 606 254_276 AD-63987.2 A-127955.9GUGGUGGACUUCUCUCAAUUU 172 A-127977.1 AAAUUGAGAGAAGUCCACCACGA 607 254_276AD-63988.2 A-127986.1 UGGACUUCUCUCAAUUU 173 A-127987.1AAAUUGAGAGAAGUCCACC 608 258_280 AD-63989.2 A-127996.1GUGGUGGACUUCUCUCAAUUU 174 A-127992.2 AAAUUGAGAGAAGUCCACCACGA 609 254_276AD-63990.2 A-127950.1 GGUGGACUUCUCUCAAUUUUU 175 A-127951.1AAAUUGAGAGAAGUCCACCUU 610 256_278 AD-63991.2 A-127962.1GUGGUGGACUUCUCUCAAUUU 176 A-127956.5 AAAUUGAGAGAAGUCCACCACGA 611 254_276AD-63992.2 A-127955.2 GUGGUGGACUUCUCUCAAUUU 177 A-127970.1AAAUUGAGAGAAGUCCACCACGA 612 254_276 AD-63993.2 A-127955.10GUGGUGGACUUCUCUCAAUUU 178 A-127978.1 AAAUUGAGAGAAGUCCACCACGA 613 254_276AD-63994.2 A-127984.2 GGUGGACUUCUCUCAAUUU 179 A-127988.1AAAUUGAGAGAAGUCCACCAC 614 256_278 AD-63995.2 A-127996.2GUGGUGGACUUCUCUCAAUUU 180 A-127993.2 AAAUUGAGAGAAGUCCACCACGA 615 254_276AD-63996.2 A-127952.1 GGUGGACUUCUCUCAAUUU 181 A-127953.1AAAUUGAGAGAAGUCCACCUU 616 256_278 AD-63997.2 A-127963.1GUGGUGGACUUCUCUCAAUUU 182 A-127956.6 AAAUUGAGAGAAGUCCACCACGA 617 254_276AD-63999.2 A-127955.11 GUGGUGGACUUCUCUCAAUUU 183 A-127979.1AAAUUGAGAGAAGUCCACCACGA 618 254_276 AD-64000.2 A-127986.2UGGACUUCUCUCAAUUU 184 A-127989.1 AAAUUGAGAGAAGUCCACC 619 258_280AD-64001.2 A-127996.3 GUGGUGGACUUCUCUCAAUUU 185 A-127994.2AAAUUGAGAGAAGUCCACCACGA 620 254_276 AD-64002.2 A-127952.2GGUGGACUUCUCUCAAUUU 186 A-127954.1 AAAUUGAGAGAAGUCCACCUU 621 256_278AD-64003.2 A-127964.1 GUGGUGGACUUCUCUCAAUUU 187 A-127956.7AAAUUGAGAGAAGUCCACCACGA 622 254_276 AD-64004.2 A-127955.4GUGGUGGACUUCUCUCAAUUU 188 A-127972.1 AAAUUGAGAGAAGUCCACCACGA 623 254_276AD-64005.2 A-127955.12 GUGGUGGACUUCUCUCAAUUU 189 A-127980.1AAAUUGAGAGAAGUCCACCACGA 624 254_276 AD-64006.2 A-127990.1GUGGUGGACUUCUCUCAAUUU 190 A-127991.1 AAAUUGAGAGAAGUCCACCACGA 625 254_276AD-64007.2 A-127996.4 GUGGUGGACUUCUCUCAAUUU 191 A-127995.2AAAUUGAGAGAAGUCCACCACGA 626 254_276 AD-64008.2 A-127955.1GUGGUGGACUUCUCUCAAUUU 192 A-127956.1 AAAUUGAGAGAAGUCCACCACGA 627 254_276AD-64008.2 A-127955.1 GUGGUGGACUUCUCUCAAUUU 193 A-127956.1AAAUUGAGAGAAGUCCACCACGA 628 254_276 AD-64008.4 A-127955.15GUGGUGGACUUCUCUCAAUUU 194 A-127956.14 AAAUUGAGAGAAGUCCACCACGA 629254_276 AD-64009.2 A-127965.1 GUGGUGGACUUCUCUCAAUUU 195 A-127956.8AAAUUGAGAGAAGUCCACCACGA 630 254_276 AD-64010.2 A-127955.5GUGGUGGACUUCUCUCAAUUU 196 A-127973.1 AAAUUGAGAGAAGUCCACCACGA 631 254_276AD-64011.2 A-127955.13 GUGGUGGACUUCUCUCAAUUU 197 A-127981.1AAAUUGAGAGAAGUCCACCACGA 632 254_276 AD-64012.2 A-127990.2GUGGUGGACUUCUCUCAAUUU 198 A-127992.1 AAAUUGAGAGAAGUCCACCACGA 633 254_276AD-64013.2 A-127997.1 GUGGUGGACTTCUCUCAAUUU 199 A-127998.1AAAUUGAGAGAAGTCCACCACGA 634 254_276 AD-64014.2 A-127957.1GUGGUGGACUUCUCUCAAUUU 200 A-127958.1 AAAUUGAGAGAAGUCCACCACGA 635 254_276AD-64015.2 A-127966.1 GUGGUGGACUUCUCUCAAUUU 201 A-127956.9AAAUUGAGAGAAGUCCACCACGA 636 254_276 AD-64016.2 A-127955.6GUGGUGGACUUCUCUCAAUUU 202 A-127974.1 AAAUUGAGAGAAGUCCACCACGA 637 254_276AD-64017.2 A-127968.2 GUGGUGGACUTCUCUCAAUUU 203 A-127982.1AAAUUGAGAGAAGTCCACCACGA 638 254_276 AD-64018.2 A-127990.3GUGGUGGACUUCUCUCAAUUU 204 A-127993.1 AAAUUGAGAGAAGUCCACCACGA 639 254_276AD-64019.2 A-127959.1 GUGGUGGACUUCUCUCAAUUU 205 A-127956.2AAAUUGAGAGAAGUCCACCACGA 640 254_276 AD-M020.2 A-127967.1GUGGUGGACUUCUCUCAAUUU 206 A-127956.10 AAAUUGAGAGAAGUCCACCACGA 641254_276 AD-M021.2 A-127955.7 GUGGUGGACUUCUCUCAAUUU 207 A-127975.1AAAUUGAGAGAAGUCCACCACGA 642 254_276 AD-M022.2 A-127968.3GUGGUGGACUTCUCUCAAUUU 208 A-127983.1 AAAUUGAGAGAAGTCCACCACGA 643 254_276AD-M023.2 A-127990.4 GUGGUGGACUUCUCUCAAUUU 209 A-127994.1AAAUUGAGAGAAGUCCACCACGA 644 254_276 AD-M024.2 A-127960.1GUGGUGGACUUCUCUCAAUUU 210 A-127956.3 AAAUUGAGAGAAGUCCACCACGA 645 254_276AD-M025.2 A-127968.1 GUGGUGGACUTCUCUCAAUUU 211 A-127956.11AAAUUGAGAGAAGUCCACCACGA 646 254_276 AD-M026.2 A-127955.8GUGGUGGACUUCUCUCAAUUU 212 A-127976.1 AAAUUGAGAGAAGUCCACCACGA 647 254_276AD-64027.2 A-127984.1 GGUGGACUUCUCUCAAUUU 213 A-127985.1AAAUUGAGAGAAGUCCACCAC 648 256_278 AD-64028.2 A-127990.5GUGGUGGACUUCUCUCAAUUU 214 A-127995.1 AAAUUGAGAGAAGUCCACCACGA 649 254_276AD-64272.2 A-128001.2 GUGCACUUCGCUUCACCUCUG 215 A-128002.2CAGAGGUGAAGCGAAGUGCACAC 650 1577_1599 AD-64274.1 A-128363.1GUUGACAAAAAUCCUCACAAU 216 A-128364.1 AUUGUGAGGAUUUUUGUCAACAA 651 215_237AD-64275.1 A-128377.1 UGUUGACAAAAAUCCUCACAA 217 A-128378.1UUGUGAGGAUUUUUGUCAACAAG 652 214_236 AD-64276.1 A-128393.1GGUGGACUUCUCUCAAUUUUA 218 A-128394.1 UAAAAUUGAGAGAAGUCCACCAC 653 256_278AD-64277.1 A-128407.1 UCUUUUGGAGUGUGGAUUCGA 219 A-128408.1UCGAAUCCACACUCCAAAAGACA 654 2259_2281 AD-64277.1 A-128407.1UCUUUUGGAGUGUGGAUUCGA 220 A-128408.1 UCGAAUCCACACUCCAAAAGACA 6552259_2281 AD-64278.1 A-128423.1 ACUGUUCAAGCCUCCAAGCUA 221 A-128424.1UAGCUUGGAGGCUUGAACAAGAC 656 1857_1879 AD-64279.1 A-128435.1UCUGCCGAUCCAUACUGCGGA 222 A-128436.1 UCCGCAGUAUGGAUCGGCAGAGG 6571255_1277 AD-64280.1 A-128379.1 AUGUGUCUGCGGCGUUUUAUA 223 A-128380.1UAUAAAACGCCGCAGACACAUCC 658 375_397 AD-64281.1 A-128395.1CCCCGUCUGUGCCUUCUCAUA 224 A-128396.1 UAUGAGAAGGCACAGACGGGGAG 6591545_1567 AD-64282.1 A-128409.1 GCCUAAUCAUCUCUUGUUCAU 225 A-128410.1AUGAACAAGAGAUGAUUAGCGAG 660 1831_1853 AD-64283.1 A-128425.1UCUAGACUCGUGGUGGACUUC 226 A-128426.1 GAAGUCCACCACGAGUCUAGACU 661 245_267AD-64284.1 A-128437.1 CUGCCGAUCCAUACUGCGGAA 227 A-128438.1UUCCGCAGUAUGGAUCGGCAGAG 662 1256_1278 AD-64285.1 A-128365.1UUUUUCUUGUUGACAAAAAUA 228 A-128366.1 UAUUUUUGUCAACAAGAAAAACC 663 207_229AD-64286.1 A-128381.1 AUCUUCUUGUUGGUUCUUCUA 229 A-128382.1UAGAAGAACCAACAAGAAGAUGA 664 426_448 AD-64289.1 A-128367.1GUUUUUCUUGUUGACAAAAAU 230 A-128368.1 AUUUUUGUCAACAAGAAAAACCC 665 206_228AD-64290.1 A-128383.1 CUGCCUAAUCAUCUCUUGUUA 231 A-128384.1UAACAAGAGAUGAUUAGGCAGAG 666 1829_1851 AD-64291.1 A-128399.1UCCUCACAAUACCACAGAGUA 232 A-128400.1 UACUCUGUGGUAUUGUGAGGAUU 667 226_248AD-64292.1 A-128413.1 CUUGUUGACAAAAAUCCUCAA 233 A-128414.1UUGAGGAUUUUUGUCAACAAGAA 668 212_234 AD-64293.1 A-128439.1GCAACUUUUUCACCUCUGCCU 234 A-128440.1 AGGCAGAGGUGAAAAAGUUGCAU 6691814_1836 AD-64294.1 A-128369.1 GGGAACAAGAGCUACAGCAUA 235 A-128370.1UAUGCUGUAGCUCUUGUUCCCAA 670 2828_2850 AD-64295.1 A-128385.1CGUGGUGGACUUCUCUCAAUU 236 A-128386.1 AAUUGAGAGAAGUCCACCAGCAG 671 253_275AD-64297.1 A-128415.1 CUGCUGCUAUGCCUCAUCUUA 237 A-128416.1UAAGAUGAGGCAUAGCAGCAGGA 672 411_433 AD-64298.1 A-128427.1GUUGGAUGUGUCUGCGGCGUU 238 A-128428.1 AACGCCGCAGACACAUCCAACGA 673 370_392AD-64299.1 A-128441.1 UUCAUCCUGCUGCUAUGCCUA 239 A-128442.1UAGGCAUAGCAGCAGGAUGAAGA 674 405_427 AD-64300.1 A-128371.1UUCUUGUUGACAAAAAUCCUA 240 A-128372.1 UAGGAUUUUUGUCAACAAGAAAA 675 210_232AD-64302.1 A-128417.1 UAUAUGGAUGAUGUGGUAUUA 241 A-128418.1UAAUACCACAUCAUCCAUAUAAC 676 734_756 AD-64303.1 A-128429.1UUCAUCCUGCUGCUAUGCCUC 242 A-128430.1 GAGGCAUAGCAGCAGGAUGAAGA 677 405_427AD-64304.1 A-128443.1 GUGCACUUCGCUUCACCUCUA 243 A-128444.1UAGAGGUGAAGCGAAGUGCACAC 678 1577_1599 AD-64305.1 A-128373.1UUGACAAAAAUCCUCACAAUA 244 A-128374.1 UAUUGUGAGGAUUUUUGUCAACA 679 216_238AD-64307.1 A-128403.1 AAGCCUCCAAGCUGUGCCUUA 245 A-128404.1UAAGGCACAGCUUGGAGGCUUGA 680 1864_1886 AD-64308.1 A-128419.1CCUCUUCAUCCUGCUGCUAUA 246 A-128420.1 UAUAGCAGCAGGAUGAAGAGGAA 681 401_423AD-64309.1 A-128431.1 CCUGCUGCUAUGCCUCAUCUU 247 A-128432.1AAGAUGAGGCAUAGCAGCAGGAU 682 410_432 AD-64310.1 A-128375.1CAUCUUCUUGUUGGUUCUUCU 248 A-128376.1 AGAAGAACCAACAAGAAGAUGAG 683 425_447AD-64311.1 A-128391.1 CCGUCUGUGCCUUCUCAUCUA 249 A-128392.1UAGAUGAGAAGGCACAGACGGGG 684 1547_1569 AD-64312.1 A-128405.1CCUCAUCUUCUUGUUGGUUCU 250 A-128406.1 AGAACCAACAAGAAGAUGAGGCA 685 422_444AD-64313.1 A-128421.1 CCACCAAAUGCCCCUAUCUUA 251 A-128422.1UAAGAUAGGGGCAUUUGGUGGUC 686 2298_2320 AD-64314.1 A-128433.1GCUCCUCUGCCGAUCCAUACU 252 A-128434.1 AGUAUGGAUCGGCAGAGGAGCCA 6871250_1272 AD-64315.1 A-128363.2 GUUGACAAAAAUCCUCACAAU 253 A-128445.1AUUGUGAGGAUUUUUGUCAACAA 688 215_237 AD-64316.1 A-128377.2UGUUGACAAAAAUCCUCACAA 254 A-128453.1 UUGUGAGGAUUUUUGUCAACAAG 689 214_236AD-64317.1 A-128393.2 GGUGGACUUCUCUCAAUUUUA 255 A-128461.1UAAAAUUGAGAGAAGUCCACCAC 690 256_278 AD-64318.1 A-128407.2UCUUUUGGAGUGUGGAUUCGA 256 A-128469.1 UCGAAUCCACACUCCAAAAGACA 6912259_2281 AD-64318.1 A-128407.2 UCUUUUGGAGUGUGGAUUCGA 257 A-128469.1UCGAAUCCACACUCCAAAAGACA 692 2259_2281 AD-64319.1 A-128423.2ACUGUUCAAGCCUCCAAGCUA 258 A-128477.1 UAGCUUGGAGGCUUGAACAAGAC 6931857_1879 AD-64320.1 A-128435.2 UCUGCCGAUCCAUACUGCGGA 259 A-128483.1UCCGCAGUAUGGAUCGGCAGAGG 694 1255_1277 AD-64321.1 A-123463.3AGUUAUAUGGAUGAUGUGGUA 260 A-128446.1 UACCACAUCAUCCAUAUAACUGA 695 731_753AD-64322.1 A-128379.2 AUGUGUCUGCGGCGUUUUAUA 261 A-128454.1UAUAAAACGCCGCAGACACAUCC 696 375_397 AD-64323.1 A-128395.2CCCCGUCUGUGCCUUCUCAUA 262 A-128462.1 UAUGAGAAGGCACAGACGGGGAG 6971545_1567 AD-64324.1 A-128409.2 GCCUAAUCAUCUCUUGUUCAU 263 A-128470.1AUGAACAAGAGAUGAUUAGCGAG 698 1831_1853 AD-64325.1 A-128425.2UCUAGACUCGUGGUGGACUUC 264 A-128478.1 GAAGUCCACCACGAGUCUAGACU 699 245_267AD-64326.1 A-128437.2 CUGCCGAUCCAUACUGCGGAA 265 A-128484.1UUCCGCAGUAUGGAUCGGCAGAG 700 1256_1278 AD-64328.1 A-128381.2AUCUUCUUGUUGGUUCUUCUA 266 A-128455.1 UAGAAGAACCAACAAGAAGAUGA 701 426_448AD-64330.1 A-128411.2 UUCUCUCAAUUUUCUAGGGGA 267 A-128471.1UCCCCUAGAAAAUUGAGAGAAGU 702 263_285 AD-64331.1 A-127905.16ACUCGUGGUGGACUUCUCUCA 268 A-127907.2 UGAGAGAAGUCCACCACGAGUCU 703 250_272AD-64332.1 A-128001.3 GUGCACUUCGCUUCACCUCUG 269 A-128485.1CAGAGGUGAAGCGAAGUGCACAC 704 1577_1599 AD-64333.1 A-128367.2GUUUUUCUUGUUGACAAAAAU 270 A-128448.1 AUUUUUGUCAACAAGAAAAACCC 705 206_228AD-64334.1 A-128383.2 CUGCCUAAUCAUCUCUUGUUA 271 A-128456.1UAACAAGAGAUGAUUAGGCAGAG 706 1829_1851 AD-64335.1 A-128399.2UCCUCACAAUACCACAGAGUA 272 A-128464.1 UACUCUGUGGUAUUGUGAGGAUU 707 226_248AD-64336.1 A-128413.2 CUUGUUGACAAAAAUCCUCAA 273 A-128472.1UUGAGGAUUUUUGUCAACAAGAA 708 212_234 AD-64337.1 A-127955.16GUGGUGGACUUCUCUCAAUUU 274 A-127958.2 AAAUUGAGAGAAGUCCACCACGA 709 254_276AD-64338.1 A-128439.2 GCAACUUUUUCACCUCUGCCU 275 A-128486.1AGGCAGAGGUGAAAAAGUUGCAU 710 1814_1836 AD-64339.1 A-128369.2GGGAACAAGAGCUACAGCAUA 276 A-128449.1 UAUGCUGUAGCUCUUGUUCCCAA 7112828_2850 AD-64341.1 A-128401.2 UCAUCUUCUUGUUGGUUCUUA 277 A-128465.1UAAGAACCAACAAGAAGAUGAGG 712 424_446 AD-64342.1 A-128415.2CUGCUGCUAUGCCUCAUCUUA 278 A-128473.1 UAAGAUGAGGCAUAGCAGCAGGA 713 411_433AD-64343.1 A-128427.2 GUUGGAUGUGUCUGCGGCGUU 279 A-128479.1AACGCCGCAGACACAUCCAACGA 714 370_392 AD-64344.1 A-128441.2UUCAUCCUGCUGCUAUGCCUA 280 A-128487.1 UAGGCAUAGCAGCAGGAUGAAGA 715 405_427AD-64345.1 A-128371.2 UUCUUGUUGACAAAAAUCCUA 281 A-128450.1UAGGAUUUUUGUCAACAAGAAAA 716 210_232 AD-64347.1 A-123487.3GGAUGUGUCUGCGGCGUUUUA 282 A-128466.1 UAAAACGCCGCAGACACAUCCAG 717 373_395AD-64348.1 A-128417.2 UAUAUGGAUGAUGUGGUAUUA 283 A-128474.1UAAUACCACAUCAUCCAUAUAAC 718 734_756 AD-64349.1 A-128429.2UUCAUCCUGCUGCUAUGCCUC 284 A-128480.1 GAGGCAUAGCAGCAGGAUGAAGA 719 405_427AD-64350.1 A-128443.2 GUGCACUUCGCUUCACCUCUA 285 A-128488.1UAGAGGUGAAGCGAAGUGCACAC 720 1577_1599 AD-64351.1 A-128373.2UUGACAAAAAUCCUCACAAUA 286 A-128451.1 UAUUGUGAGGAUUUUUGUCAACA 721 216_238AD-64352.1 A-128389.2 CCAAGUGUUUGCUGACGCAAA 287 A-128459.1UUUGCGUCAGCAAACACUUGGCA 722 1174_1196 AD-64352.1 A-128389.2CCAAGUGUUUGCUGACGCAAA 288 A-128459.1 UUUGCGUCAGCAAACACUUGGCA 7231174_1196 AD-64353.1 A-128403.2 AAGCCUCCAAGCUGUGCCUUA 289 A-128467.1UAAGGCACAGCUUGGAGGCUUGA 724 1864_1886 AD-64354.1 A-128419.2CCUCUUCAUCCUGCUGCUAUA 290 A-128475.1 UAUAGCAGCAGGAUGAAGAGGAA 725 401_423AD-64355.1 A-128431.2 CCUGCUGCUAUGCCUCAUCUU 291 A-128481.1AAGAUGAGGCAUAGCAGCAGGAU 726 410_432 AD-64356.1 A-128375.2CAUCUUCUUGUUGGUUCUUCU 292 A-128452.1 AGAAGAACCAACAAGAAGAUGAG 727 425_447AD-64357.1 A-128391.2 CCGUCUGUGCCUUCUCAUCUA 293 A-128460.1UAGAUGAGAAGGCACAGACGGGG 728 1547_1569 AD-64358.1 A-128405.2CCUCAUCUUCUUGUUGGUUCU 294 A-128468.1 AGAACCAACAAGAAGAUGAGGCA 729 422_444AD-64359.1 A-128421.2 CCACCAAAUGCCCCUAUCUUA 295 A-128476.1UAAGAUAGGGGCAUUUGGUGGUC 730 2298_2320 AD-64360.1 A-128433.2GCUCCUCUGCCGAUCCAUACU 296 A-128482.1 AGUAUGGAUCGGCAGAGGAGCCA 7311250_1272 AD-64700.1 A-129379.1 ACUCGUGGUGTACUUCUCUCA 297 A-127906.26UGAGAGAAGUCCACCACGAGUCU 732 250_272 AD-64701.1 A-127905.20ACUCGUGGUGGACUUCUCUCA 298 A-129387.1 UGAGAGAAGTCCACCACGAGUCU 733 250_272AD-64702.1 A-127905.28 ACUCGUGGUGGACUUCUCUCA 299 A-129395.1UGAGAGAAGUCCACCACGAGUCU 734 250_272 AD-64703.1 A-129376.2ACUCGUGGUGGACUUCACUCA 300 A-129385.5 UGAGAGAAGTCCACCACGAGUCU 735 250_272AD-64704.1 A-129381.3 ACUCGUGGTGTACUUCACUCA 301 A-129389.6UGAGAGAAGUCCACCACGAGUCU 736 250_272 AD-64705.1 A-129380.1ACUCGUGGUGTACUUCACUCA 302 A-127906.27 UGAGAGAAGUCCACCACGAGUCU 737250_272 AD-64706.1 A-127905.21 ACUCGUGGUGGACUUCUCUCA 303 A-129388.1UGAGAGAAGUCCACCACGAGUCU 738 250_272 AD-64707.1 A-127905.29ACUCGUGGUGGACUUCUCUCA 304 A-129396.1 UGAGAGAAGTCCACCACGAGUCU 739 250_272AD-64708.1 A-129382.2 ACUCGUGGTGGACUUCTCUCA 305 A-129385.6UGAGAGAAGTCCACCACGAGUCU 740 250_272 AD-64709.1 A-129373.4ACUCGUGGUGGACUUCUCUCA 306 A-129391.2 UGAGAGAAGTCCACCACGAGUCU 741 250_272AD-64710.1 A-129373.1 ACUCGUGGUGGACUUCUCUCA 307 A-127906.20UGAGAGAAGUCCACCACGAGUCU 742 250_272 AD-64711.1 A-129381.1ACUCGUGGTGTACUUCACUCA 308 A-127906.28 UGAGAGAAGUCCACCACGAGUCU 743250_272 AD-64712.1 A-127905.22 ACUCGUGGUGGACUUCUCUCA 309 A-129389.1UGAGAGAAGUCCACCACGAGUCU 744 250_272 AD-64713.1 A-127905.30ACUCGUGGUGGACUUCUCUCA 310 A-129397.1 UGAGAGAAGTCCACCACGAGUCU 745 250_272AD-64714.1 A-129384.2 ACUCGUGGTGGACUUCACUCA 311 A-129385.7UGAGAGAAGTCCACCACGAGUCU 746 250_272 AD-64715.1 A-129376.4ACUCGUGGUGGACUUCACUCA 312 A-129391.3 UGAGAGAAGTCCACCACGAGUCU 747 250_272AD-64716.1 A-129374.1 ACUCGUGGUGGACUUCUCUCA 313 A-127906.21UGAGAGAAGUCCACCACGAGUCU 748 250_272 AD-64717.1 A-129382.1ACUCGUGGTGGACUUCTCUCA 314 A-127906.29 UGAGAGAAGUCCACCACGAGUCU 749250_272 AD-64718.1 A-127905.23 ACUCGUGGUGGACUUCUCUCA 315 A-129390.1UGAGAGAAGUCCACCACGAGUCU 750 250_272 AD-64719.1 A-127917.5ACUCGUGGUGGACUUCTCUCA 316 A-129385.2 UGAGAGAAGTCCACCACGAGUCU 751 250_272AD-64720.1 A-129381.2 ACUCGUGGTGTACUUCACUCA 317 A-129385.8UGAGAGAAGTCCACCACGAGUCU 752 250_272 AD-64721.1 A-129382.4ACUCGUGGTGGACUUCTCUCA 318 A-129391.4 UGAGAGAAGTCCACCACGAGUCU 753 250_272AD-64722.1 A-129375.1 ACUCGUGGUGGACUUCCUCA 319 A-127906.22UGAGAGAAGUCCACCACGAGUCU 754 250_272 AD-64723.1 A-129383.1ACUCGUGGUGGACUUCTCUCA 320 A-127906.30 UGAGAGAAGUCCACCACGAGUCU 755250_272 AD-64725.1 A-127917.6 ACUCGUGGUGGACUUCTCUCA 321 A-129398.1UGAGAGAAGTCCACCACGAGUCU 756 250_272 AD-64726.1 A-129373.3ACUCGUGGUGGACUUCUCUCA 322 A-129389.2 UGAGAGAAGUCCACCACGAGUCU 757 250_272AD-64727.1 A-129384.4 ACUCGUGGTGGACUUCACUCA 323 A-129391.5UGAGAGAAGTCCACCACGAGUCU 758 250_272 AD-64728.1 A-129376.1ACUCGUGGUGGACUUCACUCA 324 A-127906.23 UGAGAGAAGUCCACCACGAGUCU 759250_272 AD-64729.1 A-129384.1 ACUCGUGGTGGACUUCACUCA 325 A-127906.31UGAGAGAAGUCCACCACGAGUCU 760 250_272 AD-64730.1 A-127905.25ACUCGUGGUGGACUUCUCUCA 326 A-129392.1 UGAGAGAAGTCCACCACGAGUCU 761 250_272AD-64731.1 A-129399.1 ACUCGUGGUGGACUUCTCUCA 327 A-129385.3UGAGAGAAGTCCACCACGAGUCU 762 250_272 AD-64732.1 A-129376.3ACUCGUGGUGGACUUCACUCA 328 A-129389.3 UGAGAGAAGUCCACCACGAGUCU 763 250_272AD-64733.1 A-129381.4 ACUCGUGGTGTACUUCACUCA 329 A-129391.6UGAGAGAAGTCCACCACGAGUCU 764 250_272 AD-64734.1 A-129377.1ACUCGUGGUGGACUUCCCUCA 330 A-127906.24 UGAGAGAAGUCCACCACGAGUCU 765250_272 AD-64735.1 A-127905.18 ACUCGUGGUGGACUUCUCUCA 331 A-129385.1UGAGAGAAGTCCACCACGAGUCU 766 250_272 AD-64736.1 A-127905.26ACUCGUGGUGGACUUCUCUCA 332 A-129393.1 UGAGAGAAGTCCACCACGAGUCU 767 250_272AD-64737.1 A-129399.2 ACUCGUGGUGGACUUCTCUCA 333 A-129398.2UGAGAGAAGTCCACCACGAGUCU 768 250_272 AD-64738.1 A-129382.3ACUCGUGGTGGACUUCTCUCA 334 A-129389.4 UGAGAGAAGUCCACCACGAGUCU 769 250_272AD-64739.1 A-129378.1 ACUCGUGGUGGACUUCGCUCA 335 A-127906.25UGAGAGAAGUCCACCACGAGUCU 770 250_272 AD-64740.1 A-127905.19ACUCGUGGUGGACUUCUCUCA 336 A-129386.1 UGAGAGAAGTCCACCACGAGUCU 771 250_272AD-64741.1 A-127905.27 ACUCGUGGUGGACUUCUCUCA 337 A-129394.1UGAGAGAAGTCCACCACGAGUCU 772 250_272 AD-64742.1 A-129373.2ACUCGUGGUGGACUUCUCUCA 338 A-129385.4 UGAGAGAAGTCCACCACGAGUCU 773 250_272AD-64743.1 A-129384.3 ACUCGUGGTGGACUUCACUCA 339 A-129389.5UGAGAGAAGUCCACCACGAGUCU 774 250_272 AD-61522.2 A-123463.2AGUUAUAUGGAUGAUGUGGUA 340 A-123464.2 UACCACAUCAUCCAUAUAACUGA 775 731_753AD-61547.2 A-123487.2 GGAUGUGUCUGCGGCGUUUUA 341 A-123488.2UAAAACGCCGCAGACACAUCCAG 776 373_395 AD-63938.2 A-127896.1ACUCGUGGUGGACUUCUCUCA 342 A-127897.1 UGAGAGAAGUCCACCACGAGUCU 777 250_272AD-63939.2 A-127909.1 ACUCGUGGUGGACUUCUCUCA 343 A-127906.3UGAGAGAAGUCCACCACGAGUCU 778 250_272 AD-63940.2 A-127917.1ACUCGUGGUGGACUUCTCUCA 344 A-127906.11 UGAGAGAAGUCCACCACGAGUCU 779250_272 AD-63941.2 A-127905.8 ACUCGUGGUGGACUUCUCUCA 345 A-127925.1UGAGAGAAGUCCACCACGAGUCU 780 250_272 AD-63942.2 A-127933.1UCGUGGUGGACUUCUCUCA 346 A-127934.1 UGAGAGAAGUCCACCACGAGU 781 252_274AD-63943.2 A-127944.2 ACUCGUGGUGGACUUCUCUCA 347 A-127942.2UGAGAGAAGUCCACCACGAGUCU 782 250_272 AD-63945.2 A-127910.1ACUCGUGGUGGACUUCUCUCA 348 A-127906.4 UGAGAGAAGUCCACCACGAGUCU 783 250_272AD-63946.2 A-127918.1 ACUCGUGGUGGACUUCUCUCA 349 A-127906.12UGAGAGAAGUCCACCACGAGUCU 784 250_272 AD-63947.2 A-127905.9ACUCGUGGUGGACUUCUCUCA 350 A-127926.1 UGAGAGAAGUCCACCACGAGUCU 785 250_272AD-63948.2 A-127935.1 GUGGUGGACUUCUCUCA 351 A-127936.1UGAGAGAAGUCCACCACGA 786 254_276 AD-63949.2 A-127944.3ACUCGUGGUGGACUUCUCUCA 352 A-127906.14 UGAGAGAAGUCCACCACGAGUCU 787250_272 AD-63950.2 A-127900.1 UCGUGGUGGACUUCUCUCAUU 353 A-127901.1UGAGAGAAGUCCACCACGAUU 788 252_274 AD-63951.2 A-127911.1ACUCGUGGUGGACUUCUCUCA 354 A-127906.5 UGAGAGAAGUCCACCACGAGUCU 789 250_272AD-63952.2 A-127905.2 ACUCGUGGUGGACUUCUCUCA 355 A-127919.1UGAGAGAAGUCCACCACGAGUCU 790 250_272 AD-63953.2 A-127905.10ACUCGUGGUGGACUUCUCUCA 356 A-127927.1 UGAGAGAAGUCCACCACGAGUCU 791 250_272AD-63955.2 A-127945.1 ACUCGUGGUGGACUUCUCUCA 357 A-127940.3UGAGAGAAGUCCACCACGAGUCU 792 250_272 AD-63956.2 A-127902.1UCGUGGUGGACUUCUCUCA 358 A-127903.1 UGAGAGAAGUCCACCACGAUU 793 252_274AD-63957.2 A-127912.1 ACUCGUGGUGGACUUCUCUCA 359 A-127906.6UGAGAGAAGUCCACCACGAGUCU 794 250_272 AD-63958.2 A-127905.3ACUCGUGGUGGACUUCUCUCA 360 A-127920.1 UGAGAGAAGUCCACCACGAGUCU 795 250_272AD-63959.2 A-127905.11 ACUCGUGGUGGACUUCUCUCA 361 A-127928.1UGAGAGAAGUCCACCACGAGUCU 796 250_272 AD-63960.2 A-126619.2UAUUUCCUAGGGUACAA 362 A-127938.1 UGAGAGAAGUCCACCACGA 797 254_276AD-63961.2 A-127945.2 ACUCGUGGUGGACUUCUCUCA 363 A-127942.3UGAGAGAAGUCCACCACGAGUCU 798 250_272 AD-63962.2 A-127902.2UCGUGGUGGACUUCUCUCA 364 A-127904.1 UGAGAGAAGUCCACCACGAUU 799 252_274AD-63963.2 A-127913.1 ACUCGUGGUGGACUUCUCUCA 365 A-127906.7UGAGAGAAGUCCACCACGAGUCU 800 250_272 AD-63964.2 A-127905.4ACUCGUGGUGGACUUCUCUCA 366 A-127921.1 UGAGAGAAGUCCACCACGAGUCU 801 250_272AD-63965.2 A-127905.12 ACUCGUGGUGGACUUCUCUCA 367 A-127929.1UGAGAGAAGUCCACCACGAGUCU 802 250_272 AD-63966.2 A-127939.1ACUCGUGGUGGACUUCUCUCA 368 A-127940.1 UGAGAGAAGUCCACCACGAGUCU 803 250_272AD-63967.2 A-127945.3 ACUCGUGGUGGACUUCUCUCA 369 A-127906.15UGAGAGAAGUCCACCACGAGUCU 804 250_272 AD-63968.2 A-127905.1ACUCGUGGUGGACUUCUCUCA 370 A-127906.1 UGAGAGAAGUCCACCACGAGUCU 805 250_272AD-63968.4 A-127905.15 ACUCGUGGUGGACUUCUCUCA 371 A-127906.17UGAGAGAAGUCCACCACGAGUCU 806 250_272 AD-63968.5 A-127905.17ACUCGUGGUGGACUUCUCUCA 372 A-127906.18 UGAGAGAAGUCCACCACGAGUCU 807250_272 AD-63969.2 A-127914.1 ACUCGUGGUGGACUUCUCUCA 373 A-127906.8UGAGAGAAGUCCACCACGAGUCU 808 250_272 AD-63970.2 A-127905.5ACUCGUGGUGGACUUCUCUCA 374 A-127922.1 UGAGAGAAGUCCACCACGAGUCU 809 250_272AD-63971.2 A-127905.13 ACUCGUGGUGGACUUCUCUCA 375 A-127930.1UGAGAGAAGUCCACCACGAGUCU 810 250_272 AD-63972.2 A-127941.1ACUCGUGGUGGACUUCUCUCA 376 A-127942.1 UGAGAGAAGUCCACCACGAGUCU 811 250_272AD-63973.2 A-127946.1 ACUCGUGGUGGACUUCUCUCA 377 A-127947.1UGAGAGAAGTCCACCACGAGUCU 812 250_272 AD-63975.2 A-127915.1ACUCGUGGUGGACUUCTCUCA 378 A-127906.9 UGAGAGAAGUCCACCACGAGUCU 813 250_272AD-63976.2 A-127905.6 ACUCGUGGUGGACUUCUCUCA 379 A-127923.1UGAGAGAAGUCCACCACGAGUCU 814 250_272 AD-63977.2 A-127917.2ACUCGUGGUGGACUUCTCUCA 380 A-127931.1 UGAGAGAAGUCCACCACGAGUCU 815 250_272AD-63978.2 A-127943.1 ACUCGUGGUGGACUUCUCUCA 381 A-127906.13UGAGAGAAGUCCACCACGAGUCU 816 250_272 AD-63979.2 A-127908.1ACUCGUGGUGGACUUCUCUCA 382 A-127906.2 UGAGAGAAGUCCACCACGAGUCU 817 250_272AD-63980.2 A-127916.1 ACUCGUGGUGGACUUCTCUCA 383 A-127906.10UGAGAGAAGUCCACCACGAGUCU 818 250_272 AD-63981.2 A-127905.7ACUCGUGGUGGACUUCUCUCA 384 A-127924.1 UGAGAGAAGUCCACCACGAGUCU 819 250_272AD-63982.2 A-127917.3 ACUCGUGGUGGACUUCTCUCA 385 A-127932.1UGAGAGAAGUCCACCACGAGUCU 820 250_272 AD-63983.2 A-127944.1ACUCGUGGUGGACUUCUCUCA 386 A-127940.2 UGAGAGAAGUCCACCACGAGUCU 821 250_272AD-63985.2 A-127961.1 GUGGUGGACUUCUCUCAAUUU 387 A-127956.4AAAUUGAGAGAAGUCCACCACGA 822 254_276 AD-63986.2 A-127969.1GUGGUGGACUUCUCUCAAUUU 388 A-127956.12 AAAUUGAGAGAAGUCCACCACGA 823254_276 AD-63987.2 A-127955.9 GUGGUGGACUUCUCUCAAUUU 389 A-127977.1AAAUUGAGAGAAGUCCACCACGA 824 254_276 AD-63988.2 A-127986.1UGGACUUCUCUCAAUUU 390 A-127987.1 AAAUUGAGAGAAGUCCACC 825 258_280AD-63989.2 A-127996.1 GUGGUGGACUUCUCUCAAUUU 391 A-127992.2AAAUUGAGAGAAGUCCACCACGA 826 254_276 AD-63990.2 A-127950.1GGUGGACUUCUCUCAAUUUUU 392 A-127951.1 AAAUUGAGAGAAGUCCACCUU 827 256_278AD-63991.2 A-127962.1 GUGGUGGACUUCUCUCAAUUU 393 A-127956.5AAAUUGAGAGAAGUCCACCACGA 828 254_276 AD-63992.2 A-127955.2GUGGUGGACUUCUCUCAAUUU 394 A-127970.1 AAAUUGAGAGAAGUCCACCACGA 829 254_276AD-63993.2 A-127955.10 GUGGUGGACUUCUCUCAAUUU 395 A-127978.1AAAUUGAGAGAAGUCCACCACGA 830 254_276 AD-63994.2 A-127984.2GGUGGACUUCUCUCAAUUU 396 A-127988.1 AAAUUGAGAGAAGUCCACCAC 831 256_278AD-63995.2 A-127996.2 GUGGUGGACUUCUCUCAAUUU 397 A-127993.2AAAUUGAGAGAAGUCCACCACGA 832 254_276 AD-63996.2 A-127952.1GGUGGACUUCUCUCAAUUU 398 A-127953.1 AAAUUGAGAGAAGUCCACCUU 833 256_278AD-63997.2 A-127963.1 GUGGUGGACUUCUCUCAAUUU 399 A-127956.6AAAUUGAGAGAAGUCCACCACGA 834 254_276 AD-63999.2 A-127955.11GUGGUGGACUUCUCUCAAUUU 400 A-127979.1 AAAUUGAGAGAAGUCCACCACGA 835 254_276AD-64000.2 A-127986.2 UGGACUUCUCUCAAUUU 401 A-127989.1AAAUUGAGAGAAGUCCACC 836 258_280 AD-64001.2 A-127996.3GUGGUGGACUUCUCUCAAUUU 402 A-127994.2 AAAUUGAGAGAAGUCCACCACGA 837 254_276AD-64002.2 A-127952.2 GGUGGACUUCUCUCAAUUU 403 A-127954.1AAAUUGAGAGAAGUCCACCUU 838 256_278 AD-64003.2 A-127964.1GUGGUGGACUUCUCUCAAUUU 404 A-127956.7 AAAUUGAGAGAAGUCCACCACGA 839 254_276AD-64004.2 A-127955.4 GUGGUGGACUUCUCUCAAUUU 405 A-127972.1AAAUUGAGAGAAGUCCACCACGA 840 254_276 AD-64005.2 A-127955.12GUGGUGGACUUCUCUCAAUUU 406 A-127980.1 AAAUUGAGAGAAGUCCACCACGA 841 254_276AD-64006.2 A-127990.1 GUGGUGGACUUCUCUCAAUUU 407 A-127991.1AAAUUGAGAGAAGUCCACCACGA 842 254_276 AD-64007.2 A-127996.4GUGGUGGACUUCUCUCAAUUU 408 A-127995.2 AAAUUGAGAGAAGUCCACCACGA 843 254_276AD-64008.2 A-127955.1 GUGGUGGACUUCUCUCAAUUU 409 A-127956.1AAAUUGAGAGAAGUCCACCACGA 844 254_276 AD-64008.4 A-127955.15GUGGUGGACUUCUCUCAAUUU 410 A-127956.14 AAAUUGAGAGAAGUCCACCACGA 845254_276 AD-64009.2 A-127965.1 GUGGUGGACUUCUCUCAAUUU 411 A-127956.8AAAUUGAGAGAAGUCCACCACGA 846 254_276 AD-64010.2 A-127955.5GUGGUGGACUUCUCUCAAUUU 412 A-127973.1 AAAUUGAGAGAAGUCCACCACGA 847 254_276AD-64011.2 A-127955.13 GUGGUGGACUUCUCUCAAUUU 413 A-127981.1AAAUUGAGAGAAGUCCACCACGA 848 254_276 AD-64012.2 A-127990.2GUGGUGGACUUCUCUCAAUUU 414 A-127992.1 AAAUUGAGAGAAGUCCACCACGA 849 254_276AD-64013.2 A-127997.1 GUGGUGGACTTCUCUCAAUUU 415 A-127998.1AAAUUGAGAGAAGTCCACCACGA 850 254_276 AD-64014.2 A-127957.1GUGGUGGACUUCUCUCAAUUU 416 A-127958.1 AAAUUGAGAGAAGUCCACCACGA 851 254_276AD-64015.2 A-127966.1 GUGGUGGACUUCUCUCAAUUU 417 A-127956.9AAAUUGAGAGAAGUCCACCACGA 852 254_276 AD-64016.2 A-127955.6GUGGUGGACUUCUCUCAAUUU 418 A-127974.1 AAAUUGAGAGAAGUCCACCACGA 853 254_276AD-64017.2 A-127968.2 GUGGUGGACUTCUCUCAAUUU 419 A-127982.1AAAUUGAGAGAAGTCCACCACGA 854 254_276 AD-64018.2 A-127990.3GUGGUGGACUUCUCUCAAUUU 420 A-127993.1 AAAUUGAGAGAAGUCCACCACGA 855 254_276AD-64019.2 A-127959.1 GUGGUGGACUUCUCUCAAUUU 421 A-127956.2AAAUUGAGAGAAGUCCACCACGA 856 254_276 AD-64020.2 A-127967.1GUGGUGGACUUCUCUCAAUUU 422 A-127956.10 AAAUUGAGAGAAGUCCACCACGA 857254_276 AD-64021.2 A-127955.7 GUGGUGGACUUCUCUCAAUUU 423 A-127975.1AAAUUGAGAGAAGUCCACCACGA 858 254_276 AD-64022.2 A-127968.3GUGGUGGACUTCUCUCAAUUU 424 A-127983.1 AAAUUGAGAGAAGTCCACCACGA 859 254_276AD-64023.2 A-127990.4 GUGGUGGACUUCUCUCAAUUU 425 A-127994.1AAAUUGAGAGAAGUCCACCACGA 860 254_276 AD-64024.2 A-127960.1GUGGUGGACUUCUCUCAAUUU 426 A-127956.3 AAAUUGAGAGAAGUCCACCACGA 861 254_276AD-64025.2 A-127968.1 GUGGUGGACUTCUCUCAAUUU 427 A-127956.11AAAUUGAGAGAAGUCCACCACGA 862 254_276 AD-64026.2 A-127955.8GUGGUGGACUUCUCUCAAUUU 428 A-127976.1 AAAUUGAGAGAAGUCCACCACGA 863 254_276AD-64027.2 A-127984.1 GGUGGACUUCUCUCAAUUU 429 A-127985.1AAAUUGAGAGAAGUCCACCAC 864 256_278 AD-64028.2 A-127990.5GUGGUGGACUUCUCUCAAUUU 430 A-127995.1 AAAUUGAGAGAAGUCCACCACGA 865 254_276AD-64272.2 A-128001.2 GUGCACUUCGCUUCACCUCUG 431 A-128002.2CAGAGGUGAAGCGAAGUGCACAC 866 1577_1599 AD-64274.1 A-128363.1GUUGACAAAAAUCCUCACAAU 432 A-128364.1 AUUGUGAGGAUUUUUGUCAACAA 867 215_237AD-64275.1 A-128377.1 UGUUGACAAAAAUCCUCACAA 433 A-128378.1UUGUGAGGAUUUUUGUCAACAAG 868 214_236 AD-64276.1 A-128393.1GGUGGACUUCUCUCAAUUUUA 434 A-128394.1 UAAAAUUGAGAGAAGUCCACCAC 869 256_278AD-64277.1 A-128407.1 UCUUUUGGAGUGUGGAUUCGA 435 A-128408.1UCGAAUCCACACUCCAAAAGACA 870 2259_2281 AD-64277.1 A-128407.1UCUUUUGGAGUGUGGAUUCGA 436 A-128408.1 UCGAAUCCACACUCCAAAAGACA 8712259_2281 AD-64278.1 A-128423.1 ACUGUUCAAGCCUCCAAGCUA 437 A-128424.1UAGCUUGGAGGCUUGAACAAGAC 872 1857_1879 AD-64279.1 A-128435.1UCUGCCGAUCCAUACUGCGGA 438 A-128436.1 UCCGCAGUAUGGAUCGGCAGAGG 8731255_1277 AD-64280.1 A-128379.1 AUGUGUCUGCGGCGUUUUAUA 439 A-128380.1UAUAAAACGCCGCAGACACAUCC 874 375_397 AD-64281.1 A-128395.1CCCCGUCUGUGCCUUCUCAUA 440 A-128396.1 UAUGAGAAGGCACAGACGGGGAG 8751545_1567 AD-64282.1 A-128409.1 GCCUAAUCAUCUCUUGUUCAU 441 A-128410.1AUGAACAAGAGAUGAUUAGCGAG 876 1831_1853 AD-64283.1 A-128425.1UCUAGACUCGUGGUGGACUUC 442 A-128426.1 GAAGUCCACCACGAGUCUAGACU 877 245_267AD-64284.1 A-128437.1 CUGCCGAUCCAUACUGCGGAA 443 A-128438.1UUCCGCAGUAUGGAUCGGCAGAG 878 1256_1278 AD-64285.1 A-128365.1UUUUUCUUGUUGACAAAAAUA 444 A-128366.1 UAUUUUUGUCAACAAGAAAAACC 879 207_229AD-64286.1 A-128381.1 AUCUUCUUGUUGGUUCUUCUA 445 A-128382.1UAGAAGAACCAACAAGAAGAUGA 880 426_448 AD-64289.1 A-128367.1GUUUUUCUUGUUGACAAAAAU 446 A-128368.1 AUUUUUGUCAACAAGAAAAACCC 881 206_228AD-64290.1 A-128383.1 CUGCCUAAUCAUCUCUUGUUA 447 A-128384.1UAACAAGAGAUGAUUAGGCAGAG 882 1829_1851 AD-64291.1 A-128399.1UCCUCACAAUACCACAGAGUA 448 A-128400.1 UACUCUGUGGUAUUGUGAGGAUU 883 226_248AD-64292.1 A-128413.1 CUUGUUGACAAAAAUCCUCAA 449 A-128414.1UUGAGGAUUUUUGUCAACAAGAA 884 212_234 AD-64293.1 A-128439.1GCAACUUUUUCACCUCUGCCU 450 A-128440.1 AGGCAGAGGUGAAAAAGUUGCAU 8851814_1836 AD-64294.1 A-128369.1 GGGAACAAGAGCUACAGCAUA 451 A-128370.1UAUGCUGUAGCUCUUGUUCCCAA 886 2828_2850 AD-64295.1 A-128385.1CGUGGUGGACUUCUCUCAAUU 452 A-128386.1 AAUUGAGAGAAGUCCACCAGCAG 887 253_275AD-64297.1 A-128415.1 CUGCUGCUAUGCCUCAUCUUA 453 A-128416.1UAAGAUGAGGCAUAGCAGCAGGA 888 411_433 AD-64298.1 A-128427.1GUUGGAUGUGUCUGCGGCGUU 454 A-128428.1 AACGCCGCAGACACAUCCAACGA 889 370_392AD-64299.1 A-128441.1 UUCAUCCUGCUGCUAUGCCUA 455 A-128442.1UAGGCAUAGCAGCAGGAUGAAGA 890 405_427 AD-64300.1 A-128371.1UUCUUGUUGACAAAAAUCCUA 456 A-128372.1 UAGGAUUUUUGUCAACAAGAAAA 891 210_232AD-64302.1 A-128417.1 UAUAUGGAUGAUGUGGUAUUA 457 A-128418.1UAAUACCACAUCAUCCAUAUAAC 892 734_756 AD-64303.1 A-128429.1UUCAUCCUGCUGCUAUGCCUC 458 A-128430.1 GAGGCAUAGCAGCAGGAUGAAGA 893 405_427AD-64304.1 A-128443.1 GUGCACUUCGCUUCACCUCUA 459 A-128444.1UAGAGGUGAAGCGAAGUGCACAC 894 1577_1599 AD-64305.1 A-128373.1UUGACAAAAAUCCUCACAAUA 460 A-128374.1 UAUUGUGAGGAUUUUUGUCAACA 895 216_238AD-64307.1 A-128403.1 AAGCCUCCAAGCUGUGCCUUA 461 A-128404.1UAAGGCACAGCUUGGAGGCUUGA 896 1864_1886 AD-64308.1 A-128419.1CCUCUUCAUCCUGCUGCUAUA 462 A-128420.1 UAUAGCAGCAGGAUGAAGAGGAA 897 401_423AD-64309.1 A-128431.1 CCUGCUGCUAUGCCUCAUCUU 463 A-128432.1AAGAUGAGGCAUAGCAGCAGGAU 898 410_432 AD-64310.1 A-128375.1CAUCUUCUUGUUGGUUCUUCU 464 A-128376.1 AGAAGAACCAACAAGAAGAUGAG 899 425_447AD-64311.1 A-128391.1 CCGUCUGUGCCUUCUCAUCUA 465 A-128392.1UAGAUGAGAAGGCACAGACGGGG 900 1547_1569 AD-64312.1 A-128405.1CCUCAUCUUCUUGUUGGUUCU 466 A-128406.1 AGAACCAACAAGAAGAUGAGGCA 901 422_444AD-64313.1 A-128421.1 CCACCAAAUGCCCCUAUCUUA 467 A-128422.1UAAGAUAGGGGCAUUUGGUGGUC 902 2298_2320 AD-64314.1 A-128433.1GCUCCUCUGCCGAUCCAUACU 468 A-128434.1 AGUAUGGAUCGGCAGAGGAGCCA 9031250_1272 AD-64315.1 A-128363.2 GUUGACAAAAAUCCUCACAAU 469 A-128445.1AUUGUGAGGAUUUUUGUCAACAA 904 215_237 AD-64316.1 A-128377.2UGUUGACAAAAAUCCUCACAA 470 A-128453.1 UUGUGAGGAUUUUUGUCAACAAG 905 214_236AD-64317.1 A-128393.2 GGUGGACUUCUCUCAAUUUUA 471 A-128461.1UAAAAUUGAGAGAAGUCCACCAC 906 256_278 AD-64318.1 A-128407.2UCUUUUGGAGUGUGGAUUCGA 472 A-128469.1 UCGAAUCCACACUCCAAAAGACA 9072259_2281 AD-64318.1 A-128407.2 UCUUUUGGAGUGUGGAUUCGA 473 A-128469.1UCGAAUCCACACUCCAAAAGACA 908 2259_2281 AD-64319.1 A-128423.2ACUGUUCAAGCCUCCAAGCUA 474 A-128477.1 UAGCUUGGAGGCUUGAACAAGAC 9091857_1879 AD-64320.1 A-128435.2 UCUGCCGAUCCAUACUGCGGA 475 A-128483.1UCCGCAGUAUGGAUCGGCAGAGG 910 1255_1277 AD-64321.1 A-123463.3AGUUAUAUGGAUGAUGUGGUA 476 A-128446.1 UACCACAUCAUCCAUAUAACUGA 911 731_753AD-64322.1 A-128379.2 AUGUGUCUGCGGCGUUUUAUA 477 A-128454.1UAUAAAACGCCGCAGACACAUCC 912 375_397 AD-64323.1 A-128395.2CCCCGUCUGUGCCUUCUCAUA 478 A-128462.1 UAUGAGAAGGCACAGACGGGGAG 9131545_1567 AD-64324.1 A-128409.2 GCCUAAUCAUCUCUUGUUCAU 479 A-128470.1AUGAACAAGAGAUGAUUAGCGAG 914 1831_1853 AD-64325.1 A-128425.2UCUAGACUCGUGGUGGACUUC 480 A-128478.1 GAAGUCCACCACGAGUCUAGACU 915 245_267AD-64326.1 A-128437.2 CUGCCGAUCCAUACUGCGGAA 481 A-128484.1UUCCGCAGUAUGGAUCGGCAGAG 916 1256_1278 AD-64328.1 A-128381.2AUCUUCUUGUUGGUUCUUCUA 482 A-128455.1 UAGAAGAACCAACAAGAAGAUGA 917 426_448AD-64330.1 A-128411.2 UUCUCUCAAUUUUCUAGGGGA 483 A-128471.1UCCCCUAGAAAAUUGAGAGAAGU 918 263_285 AD-64331.1 A-127905.16ACUCGUGGUGGACUUCUCUCA 484 A-127907.2 UGAGAGAAGUCCACCACGAGUCU 919 250_272AD-64332.1 A-128001.3 GUGCACUUCGCUUCACCUCUG 485 A-128485.1CAGAGGUGAAGCGAAGUGCACAC 920 1577_1599 AD-64333.1 A-128367.2GUUUUUCUUGUUGACAAAAAU 486 A-128448.1 AUUUUUGUCAACAAGAAAAACCC 921 206_228AD-64334.1 A-128383.2 CUGCCUAAUCAUCUCUUGUUA 487 A-128456.1UAACAAGAGAUGAUUAGGCAGAG 922 1829_1851 AD-64335.1 A-128399.2UCCUCACAAUACCACAGAGUA 488 A-128464.1 UACUCUGUGGUAUUGUGAGGAUU 923 226_248AD-64336.1 A-128413.2 CUUGUUGACAAAAAUCCUCAA 489 A-128472.1UUGAGGAUUUUUGUCAACAAGAA 924 212_234 AD-64337.1 A-127955.16GUGGUGGACUUCUCUCAAUUU 490 A-127958.2 AAAUUGAGAGAAGUCCACCACGA 925 254_276AD-64338.1 A-128439.2 GCAACUUUUUCACCUCUGCCU 491 A-128486.1AGGCAGAGGUGAAAAAGUUGCAU 926 1814_1836 AD-64339.1 A-128369.2GGGAACAAGAGCUACAGCAUA 492 A-128449.1 UAUGCUGUAGCUCUUGUUCCCAA 9272828_2850 AD-64341.1 A-128401.2 UCAUCUUCUUGUUGGUUCUUA 493 A-128465.1UAAGAACCAACAAGAAGAUGAGG 928 424_446 AD-64342.1 A-128415.2CUGCUGCUAUGCCUCAUCUUA 494 A-128473.1 UAAGAUGAGGCAUAGCAGCAGGA 929 411_433AD-64343.1 A-128427.2 GUUGGAUGUGUCUGCGGCGUU 495 A-128479.1AACGCCGCAGACACAUCCAACGA 930 370_392 AD-64344.1 A-128441.2UUCAUCCUGCUGCUAUGCCUA 496 A-128487.1 UAGGCAUAGCAGCAGGAUGAAGA 931 405_427AD-64345.1 A-128371.2 UUCUUGUUGACAAAAAUCCUA 497 A-128450.1UAGGAUUUUUGUCAACAAGAAAA 932 210_232 AD-64347.1 A-123487.3GGAUGUGUCUGCGGCGUUUUA 498 A-128466.1 UAAAACGCCGCAGACACAUCCAG 933 373_395AD-64348.1 A-128417.2 UAUAUGGAUGAUGUGGUAUUA 499 A-128474.1UAAUACCACAUCAUCCAUAUAAC 934 734_756 AD-64349.1 A-128429.2UUCAUCCUGCUGCUAUGCCUC 500 A-128480.1 GAGGCAUAGCAGCAGGAUGAAGA 935 405_427AD-64350.1 A-128443.2 GUGCACUUCGCUUCACCUCUA 501 A-128488.1UAGAGGUGAAGCGAAGUGCACAC 936 1577_1599 AD-64351.1 A-128373.2UUGACAAAAAUCCUCACAAUA 502 A-128451.1 UAUUGUGAGGAUUUUUGUCAACA 937 216_238AD-64352.1 A-128389.2 CCAAGUGUUUGCUGACGCAAA 503 A-128459.1UUUGCGUCAGCAAACACUUGGCA 938 1174_1196 AD-64352.1 A-128389.2CCAAGUGUUUGCUGACGCAAA 504 A-128459.1 UUUGCGUCAGCAAACACUUGGCA 9391174_1196 AD-64353.1 A-128403.2 AAGCCUCCAAGCUGUGCCUUA 505 A-128467.1UAAGGCACAGCUUGGAGGCUUGA 940 1864_1886 AD-64354.1 A-128419.2CCUCUUCAUCCUGCUGCUAUA 506 A-128475.1 UAUAGCAGCAGGAUGAAGAGGAA 941 401_423AD-64355.1 A-128431.2 CCUGCUGCUAUGCCUCAUCUU 507 A-128481.1AAGAUGAGGCAUAGCAGCAGGAU 942 410_432 AD-64356.1 A-128375.2CAUCUUCUUGUUGGUUCUUCU 508 A-128452.1 AGAAGAACCAACAAGAAGAUGAG 943 425_447AD-64357.1 A-128391.2 CCGUCUGUGCCUUCUCAUCUA 509 A-128460.1UAGAUGAGAAGGCACAGACGGGG 944 1547_1569 AD-64358.1 A-128405.2CCUCAUCUUCUUGUUGGUUCU 510 A-128468.1 AGAACCAACAAGAAGAUGAGGCA 945 422_444AD-64359.1 A-128421.2 CCACCAAAUGCCCCUAUCUUA 511 A-128476.1UAAGAUAGGGGCAUUUGGUGGUC 946 2298_2320 AD-64360.1 A-128433.2GCUCCUCUGCCGAUCCAUACU 512 A-128482.1 AGUAUGGAUCGGCAGAGGAGCCA 9471250_1272 AD-64700.1 A-129379.1 ACUCGUGGUGTACUUCUCUCA 513 A-127906.26UGAGAGAAGUCCACCACGAGUCU 948 250_272 AD-64701.1 A-127905.20ACUCGUGGUGGACUUCUCUCA 514 A-129387.1 UGAGAGAAGTCCACCACGAGUCU 949 250_272AD-64702.1 A-127905.28 ACUCGUGGUGGACUUCUCUCA 515 A-129395.1UGAGAGAAGUCCACCACGAGUCU 950 250_272 AD-64703.1 A-129376.2ACUCGUGGUGGACUUCACUCA 516 A-129385.5 UGAGAGAAGTCCACCACGAGUCU 951 250_272AD-64704.1 A-129381.3 ACUCGUGGTGTACUUCACUCA 517 A-129389.6UGAGAGAAGUCCACCACGAGUCU 952 250_272 AD-64705.1 A-129380.1ACUCGUGGUGTACUUCACUCA 518 A-127906.27 UGAGAGAAGUCCACCACGAGUCU 953250_272 AD-64706.1 A-127905.21 ACUCGUGGUGGACUUCUCUCA 519 A-129388.1UGAGAGAAGUCCACCACGAGUCU 954 250_272 AD-64707.1 A-127905.29ACUCGUGGUGGACUUCUCUCA 520 A-129396.1 UGAGAGAAGTCCACCACGAGUCU 955 250_272AD-64708.1 A-129382.2 ACUCGUGGTGGACUUCTCUCA 521 A-129385.6UGAGAGAAGTCCACCACGAGUCU 956 250_272 AD-64709.1 A-129373.4ACUCGUGGUGGACUUCUCUCA 522 A-129391.2 UGAGAGAAGTCCACCACGAGUCU 957 250_272AD-64710.1 A-129373.1 ACUCGUGGUGGACUUCUCUCA 523 A-127906.20UGAGAGAAGUCCACCACGAGUCU 958 250_272 AD-64711.1 A-129381.1ACUCGUGGTGTACUUCACUCA 524 A-127906.28 UGAGAGAAGUCCACCACGAGUCU 959250_272 AD-64712.1 A-127905.22 ACUCGUGGUGGACUUCUCUCA 525 A-129389.1UGAGAGAAGUCCACCACGAGUCU 960 250_272 AD-64713.1 A-127905.30ACUCGUGGUGGACUUCUCUCA 526 A-129397.1 UGAGAGAAGTCCACCACGAGUCU 961 250_272AD-64714.1 A-129384.2 ACUCGUGGTGGACUUCACUCA 527 A-129385.7UGAGAGAAGTCCACCACGAGUCU 962 250_272 AD-64715.1 A-129376.4ACUCGUGGUGGACUUCACUCA 528 A-129391.3 UGAGAGAAGTCCACCACGAGUCU 963 250_272AD-64716.1 A-129374.1 ACUCGUGGUGGACUUCUCUCA 529 A-127906.21UGAGAGAAGUCCACCACGAGUCU 964 250_272 AD-64717.1 A-129382.1ACUCGUGGTGGACUUCTCUCA 530 A-127906.29 UGAGAGAAGUCCACCACGAGUCU 965250_272 AD-64718.1 A-127905.23 ACUCGUGGUGGACUUCUCUCA 531 A-129390.1UGAGAGAAGUCCACCACGAGUCU 966 250_272 AD-64719.1 A-127917.5ACUCGUGGUGGACUUCTCUCA 532 A-129385.2 UGAGAGAAGTCCACCACGAGUCU 967 250_272AD-64720.1 A-129381.2 ACUCGUGGTGTACUUCACUCA 533 A-129385.8UGAGAGAAGTCCACCACGAGUCU 968 250_272 AD-64721.1 A-129382.4ACUCGUGGTGGACUUCTCUCA 534 A-129391.4 UGAGAGAAGTCCACCACGAGUCU 969 250_272AD-64722.1 A-129375.1 ACUCGUGGUGGACUUCCUCA 535 A-127906.22UGAGAGAAGUCCACCACGAGUCU 970 250_272 AD-64723.1 A-129383.1ACUCGUGGUGGACUUCTCUCA 536 A-127906.30 UGAGAGAAGUCCACCACGAGUCU 971250_272 AD-64725.1 A-127917.6 ACUCGUGGUGGACUUCTCUCA 537 A-129398.1UGAGAGAAGTCCACCACGAGUCU 972 250_272 AD-64726.1 A-129373.3ACUCGUGGUGGACUUCUCUCA 538 A-129389.2 UGAGAGAAGUCCACCACGAGUCU 973 250_272AD-64727.1 A-129384.4 ACUCGUGGTGGACUUCACUCA 539 A-129391.5UGAGAGAAGTCCACCACGAGUCU 974 250_272 AD-64728.1 A-129376.1ACUCGUGGUGGACUUCACUCA 540 A-127906.23 UGAGAGAAGUCCACCACGAGUCU 975250_272 AD-64729.1 A-129384.1 ACUCGUGGTGGACUUCACUCA 541 A-127906.31UGAGAGAAGUCCACCACGAGUCU 976 250_272 AD-64730.1 A-127905.25ACUCGUGGUGGACUUCUCUCA 542 A-129392.1 UGAGAGAAGTCCACCACGAGUCU 977 250_272AD-64731.1 A-129399.1 ACUCGUGGUGGACUUCTCUCA 543 A-129385.3UGAGAGAAGTCCACCACGAGUCU 978 250_272 AD-64732.1 A-129376.3ACUCGUGGUGGACUUCACUCA 544 A-129389.3 UGAGAGAAGUCCACCACGAGUCU 979 250_272AD-64733.1 A-129381.4 ACUCGUGGTGTACUUCACUCA 545 A-129391.6UGAGAGAAGTCCACCACGAGUCU 980 250_272 AD-64734.1 A-129377.1ACUCGUGGUGGACUUCCCUCA 546 A-127906.24 UGAGAGAAGUCCACCACGAGUCU 981250_272 AD-64735.1 A-127905.18 ACUCGUGGUGGACUUCUCUCA 547 A-129385.1UGAGAGAAGTCCACCACGAGUCU 982 250_272 AD-64736.1 A-127905.26ACUCGUGGUGGACUUCUCUCA 548 A-129393.1 UGAGAGAAGTCCACCACGAGUCU 983 250_272AD-64737.1 A-129399.2 ACUCGUGGUGGACUUCTCUCA 549 A-129398.2UGAGAGAAGTCCACCACGAGUCU 984 250_272 AD-64738.1 A-129382.3ACUCGUGGTGGACUUCTCUCA 550 A-129389.4 UGAGAGAAGUCCACCACGAGUCU 985 250_272AD-64739.1 A-129378.1 ACUCGUGGUGGACUUCGCUCA 551 A-127906.25UGAGAGAAGUCCACCACGAGUCU 986 250_272 AD-64740.1 A-127905.19ACUCGUGGUGGACUUCUCUCA 552 A-129386.1 UGAGAGAAGTCCACCACGAGUCU 987 250_272AD-64741.1 A-127905.27 ACUCGUGGUGGACUUCUCUCA 553 A-129394.1UGAGAGAAGTCCACCACGAGUCU 988 250_272 AD-64742.1 A-129373.2ACUCGUGGUGGACUUCUCUCA 554 A-129385.4 UGAGAGAAGTCCACCACGAGUCU 989 250_272AD-64743.1 A-129384.3 ACUCGUGGTGGACUUCACUCA 555 A-129389.5UGAGAGAAGUCCACCACGAGUCU 990 250_272

TABLE 4 Modified Sense and Antisense Strand Sequences of HBV dsRNAs SEQSEQ Duplex Sense Oligo ID Antisense Antisense Sequence ID Name NameSense Sequence (5′ to 3′) NO: Oligo Name (5′ to 3′) NO: AD-61522.2A-123463.2 AfsgsUfuAfuAfuGfGfAfuGfaU  991 A-123464.2usAfscCfaCfaUfcAfuccAfuA 1210 fgUfgGfuAfL96 fuAfaCfusgsa AD-61547.2A-123487.2 GfsgsAfuGfuGfuCfUfGfcGfgC  992 A-123488.2usAfsaAfaCfgCfcGfcagAfcA 1211 fgUfuUfuAfL96 fcAfuCfcsasg AD-63938.2A-127896.1 Y44ACUCGUGGUGGACUUCUCUCA  993 A-127897.1UGAGAGAAGUCCACCACGAGUCU 1212 AD-63939.2 A-127909.1ascsucGfuGfgUfGfGfaCfuucU  994 A-127906.3 usGfsaGfaGfaAfgUfccaCfcA 1213fcucaL96 fcGfaGfuscsu AD-63940.2 A-127917.1 ascsucguggugdGacuuc(Tgn)c 995 A-127906.11 usGfsaGfaGfaAfgUfccaCfcA 1214 ucaL96 fcGfaGfuscsuAD-63940.3 A-127917.4 ascsucguggugdGacuuc(Tgn)c  996 A-127906.19usGfsaGfaGfaAfgUfccaCfcA 1215 ucaL96 fcGfaGfuscsu AD-63941.2 A-127905.8AfscsUfcGfuGfgUfGfGfaCfuU  997 A-127925.1 usGfsaGfagaAfguccaCfcAfc 1216fcUfcUfcAfL96 gaGfuscsu AD-63942.2 A-127933.1 uscsGfuGfgUfGfGfaCfuUfcUf 998 A-127934.1 usGfsaGfaGfaAfgUfccaCfcA 1217 cUfcAfL96 fcGfasgsuAD-63943.2 A-127944.2 ascsucGfuGfguGfGfaCfuucuc  999 A-127942.2usGfsAfgaGfaAfgUfccaCfcA 1218 ucaL96 fcGfaguscsu AD-63945.2 A-127910.1ascsucguGfgUfGfGfaCfuucUf 1000 A-127906.4 usGfsaGfaGfaAfgUfccaCfcA 1219cucaL96 fcGfaGfuscsu AD-63946.2 A-127918.1 ascsucguGfgUfGfGfacuuCfuc1001 A-127906.12 usGfsaGfaGfaAfgUfccaCfcA 1220 ucaL96 fcGfaGfuscsuAD-63947.2 A-127905.9 AfscsUfcGfuGfgUfGfGfaCfuU 1002 A-127926.1usGfsaGfagaagUfccaCfcAfc 1221 fcUfcUfcAfL96 gaGfuscsu AD-63948.2A-127935.1 gsusGfgUfGfGfaCfuUfcUfcUf 1003 A-127936.1usGfsaGfaGfaAfgUfccaCfcA 1222 cAfL96 fcsgsa AD-63949.2 A-127944.3ascsucGfuGfguGfGfaCfuucuc 1004 A-127906.14 usGfsaGfaGfaAfgUfccaCfcA 1223ucaL96 fcGfaGfuscsu AD-63950.2 A-127900.1 Y44UfcGfuGfgUfgGfaCfuUfcU 1005A-127901.1 usGfsasGfaGfaAfgUfcCfaCf 1224 fcUfcAfusuY44 cAfcGfausuAD-63951.2 A-127911.1 ascsucguGfgUfGfGfaCfuucuc 1006 A-127906.5usGfsaGfaGfaAfgUfccaCfcA 1225 ucaL96 fcGfaGfuscsu AD-63952.2 A-127905.2AfscsUfcGfuGfgUfGfGfaCfuU 1007 A-127919.1 usGfsaGfaGfaagUfccaCfcAf 1226fcUfcUfcAfL96 cGfaGfuscsu AD-63953.2 A-127905.10AfscsUfcGfuGfgUfGfGfaCfuU 1008 A-127927.1 usGfsagagaAfgUfccaCfcAfc 1227fcUfcUfcAfL96 gaguscsu AD-63955.2 A-127945.1 ascsucgugguGfGfacuucucuca1009 A-127940.3 usGfsAfgAfgAfaGfuccaCfCf 1228 L96 aCfgAfguscsuAD-63956.2 A-127902.1 Y44uscsGfuGfgUfgGfaCfuUfc 1010 A-127903.1usGfsaGfaGfaAfgUfcCfaCfc 1229 UfcUfcAfY44 AfcGfasusu AD-63957.2A-127912.1 ascsucguGfgUfGfGfacuucucu 1011 A-127906.6usGfsaGfaGfaAfgUfccaCfcA 1230 caL96 fcGfaGfuscsu AD-63958.2 A-127905.3AfscsUfcGfuGfgUfGfGfaCfuU 1012 A-127920.1 usGfsagaGfaAfgUfccaCfcAf 1231fcUfcUfcAfL96 cgaGfuscsu AD-63959.2 A-127905.11AfscsUfcGfuGfgUfGfGfaCfuU 1013 A-127928.1 usGfsaGfagaAfguccaCfcAfc 1232fcUfcUfcAfL96 gaguscsu AD-63960.2 A-126619.2 usasUfuUfCfCfuAfgGfgUfaCf1014 A-127938.1 PusGfsaGfaGfaAfgUfccaCfc 1233 aAfL96 Afcsgsa AD-63961.2A-127945.2 ascsucgugguGfGfacuucucuca 1015 A-127942.3usGfsAfgaGfaAfgUfccaCfcA 1234 L96 fcGfaguscsu AD-63962.2 A-127902.2Y44uscsGfuGfgUfgGfaCfuUfc 1016 A-127904.1 PusGfsaGfaGfaAfgUfcCfaCf 1235UfcUfcAfY44 cAfcGfasusu AD-63963.2 A-127913.1 ascsucguggUfgGfacuucucuca1017 A-127906.7 usGfsaGfaGfaAfgUfccaCfcA 1236 L96 fcGfaGfuscsuAD-63964.2 A-127905.4 AfscsUfcGfuGfgUfGfGfaCfuU 1018 A-127921.1usGfsaGfaGfaAfgUfccaCfcA 1237 fcUfcUfcAfL96 fcgaguscsu AD-63965.2A-127905.12 AfscsUfcGfuGfgUfGfGfaCfuU 1019 A-127929.1usGfsagaGfaaGfuccaCfcAfc 1238 fcUfcUfcAfL96 gaguscsu AD-63966.2A-127939.1 ascsUfcGfugguGfGfaCfuuCfu 1020 A-127940.1usGfsAfgAfgAfaGfuccaCfCf 1239 CfucaL96 aCfgAfguscsu AD-63967.2A-127945.3 ascsucgugguGfGfacuucucuca 1021 A-127906.15usGfsaGfaGfaAfgUfccaCfcA 1240 L96 fcGfaGfuscsu AD-63968.2 A-127905.1AfscsUfcGfuGfgUfGfGfaCfuU 1022 A-127906.1 usGfsaGfaGfaAfgUfccaCfcA 1241fcUfcUfcAfL96 fcGfaGfuscsu AD-63968.2 A-127905.1AfscsUfcGfuGfgUfGfGfaCfuU 1023 A-127906.1 usGfsaGfaGfaAfgUfccaCfcA 1242fcUfcUfcAfL96 fcGfaGfuscsu AD-63968.4 A-127905.15AfscsUfcGfuGfgUfGfGfaCfuU 1024 A-127906.17 usGfsaGfaGfaAfgUfccaCfcA 1243fcUfcUfcAfL96 fcGfaGfuscsu AD-63968.5 A-127905.17AfscsUfcGfuGfgUfGfGfaCfuU 1025 A-127906.18 usGfsaGfaGfaAfgUfccaCfcA 1244fcUfcUfcAfL96 fcGfaGfuscsu AD-63969.2 A-127914.1ascsucguggugGfacuucucucaL 1026 A-127906.8 usGfsaGfaGfaAfgUfccaCfcA 124596 fcGfaGfuscsu AD-63970.2 A-127905.5 AfscsUfcGfuGfgUfGfGfaCfuU 1027A-127922.1 usGfsagaGfaagUfccaCfcAfc 1246 fcUfcUfcAfL96 gaGfuscsuAD-63971.2 A-127905.13 AfscsUfcGfuGfgUfGfGfaCfuU 1028 A-127930.1usGfsagaGfaaguccaCfcAfcg 1247 fcUfcUfcAfL96 aguscsu AD-63972.2A-127941.1 ascsUfcGfuGfguGfGfaCfuuCf 1029 A-127942.1usGfsAfgaGfaAfgUfccaCfcA 1248 uCfucaL96 fcGfaguscsu AD-63973.2A-127946.1 ascsucguggudGdGacuucucuca 1030 A-127947.1usdGsaGfaGfaAfgdTccadCcA 1249 L96 fcGfaguscsu AD-63975.2 A-127915.1ascsucguggUfgGfacuuc(Tgn) 1031 A-127906.9 usGfsaGfaGfaAfgUfccaCfcA 1250cucaL96 fcGfaGfuscsu AD-63976.2 A-127905.6 AfscsUfcGfuGfgUfGfGfaCfuU1032 A-127923.1 usGfsagaGfaAfgUfccaCfcAf 1251 fcUfcUfcAfL96 cgaguscsuAD-63977.2 A-127917.2 ascsucguggugdGacuuc(Tgn)c 1033 A-127931.1usdGsagagaaguccadCcacgag 1252 ucaL96 uscsu AD-63978.2 A-127943.1ascsUfcGfuGfguGfGfaCfuUfc 1034 A-127906.13 usGfsaGfaGfaAfgUfccaCfcA 1253UfcUfcaL96 fcGfaGfuscsu AD-63979.2 A-127908.1 ascsucGfuGfgUfGfGfaCfuucU1035 A-127906.2 usGfsaGfaGfaAfgUfccaCfcA 1254 fcucAfL96 fcGfaGfuscsuAD-63980.2 A-127916.1 ascsucguggugGfacuuc(Tgn)c 1036 A-127906.10usGfsaGfaGfaAfgUfccaCfcA 1255 ucaL96 fcGfaGfuscsu AD-63981.2 A-127905.7AfscsUfcGfuGfgUfGfGfaCfuU 1037 A-127924.1 usGfsaGfagaAfgUfccaCfcAf 1256fcUfcUfcAfL96 cgaGfuscsu AD-63982.2 A-127917.3 ascsucguggugdGacuuc(Tgn)c1038 A-127932.1 PusdGsagagaaguccadCcacga 1257 ucaL96 guscsu AD-63983.2A-127944.1 ascsucGfuGfguGfGfaCfuucuc 1039 A-127940.2usGfsAfgAfgAfaGfuccaCfCf 1258 ucaL96 aCfgAfguscsu AD-63985.2 A-127961.1gsusggugGfaCfUfUfcUfcucAf 1040 A-127956.4 asAfsaUfuGfaGfaGfaagUfcC 1259auuuL96 faCfcAfcsgsa AD-63986.2 A-127969.1 gsusggugGfaCfUfUfcucuCfaa1041 A-127956.12 asAfsaUfuGfaGfaGfaagUfcC 1260 uuuL96 faCfcAfcsgsaAD-63987.2 A-127955.9 GfsusGfgUfgGfaCfUfUfcUfcU 1042 A-127977.1asAfsaUfugagaGfaagUfcCfa 1261 fcAfaUfaUfL96 ccAfcsgsa AD-63988.2A-127986.1 usgsGfaCfUfUfcUlcUfcAfaUf 1043 A-127987.1asAfsaUfuGfaGfaGfaagUfcC 1262 aUfL96 fascsc AD-63989.2 A-127996.1gsusgguggacUfUfcucucaauuu 1044 A-127992.2 asAfsAfUfuGfaGfaGfaagUfc 1263L96 CfaCfcacsgsa AD-63990.2 A-127950.1 Y44GfgUfgGfaCfuUfcUfcUfcA 1045A-127951.1 asAfsasUfuGfaGfaGfaAfgUf 1264 faUfuUfusuY44 cCfaCfcusuAD-63991.2 A-127962.1 gsusggugGfaCfUfUfcUfcucaa 1046 A-127956.5asAfsaUfuGfaGfaGfaagUfcC 1265 uuuL96 faCfcAfcsgsa AD-63992.2 A-127955.2GfsusGfgUfgGfaCfUfUfcUfcU 1047 A-127970.1 asAfsaUfuGfagaGfaagUfcCf 1266fcAfaUfuUfL96 aCfcAfcsgsa AD-63993.2 A-127955.10GfsusGfgUfgGfaCfUfUfcUfcU 1048 A-127978.1 asAfsauugaGfaGfaagUfcCfa 1267fcAfaUfuUfL96 acccsgsa AD-63994.2 A-127984.2 gsgUfgGfaCfUfUfcUfcUfcAfa1049 A-127988.1 PasAfsaUfuGfaGfaGfaagUfc 1268 UfuUfL96 CfaCfcsascAD-63995.2 A-127996.2 gsusgguggacUfUfcucucaauuu 1050 A-127993.2asAfsAfuuGfaGfaGfaagUfCf 1269 L96 caCfcacsgsa AD-63996.2 A-127952.1Y44gsgsUfgGfaCfuUfcUfcUfc 1051 A-127953.1 asAfsaUfuGfaGfaGfaAfgUfc 1270AfaUfuUfY44 CfaCfcsusu AD-63997.2 A-127963.1 gsusggugGfaCfUfUfcucucaau1052 A-127956.6 asAfsaUfuGfaGfaGfaagUfcC 1271 uuL96 faCfcAfcsgsaAD-63999.2 A-127955.11 GfsusGfgUfgGfaCfUfUfcUfcU 1053 A-127979.1asAfsaUfugaGfagaagUfcCfa 1272 fcAfaUfuUfL96 ccacsgsa AD-64000.2A-127986.2 usgsGfaCfUfUfcUfcUfcAfaUf 1054 A-127989.1PasAfsaUfuGfaGfaGfaagUfc 1273 uUfL96 Cfascsc AD-64001.2 A-127996.3gsusgguggacUfUfcucucaauuu 1055 A-127994.2 asAfsAfUfuGfaGfaGfaagUfC 1274L96 fcaCfcacsgsa AD-64002.2 A-127952.2 Y44gsgsUfgGfaCfuUfcUfcUfc 1056A-127954.1 PasAfsaUfuGfaGfaGfaAfgUf 1275 AfaUfuUfY44 cCfaCfcsusuAD-64003.2 A-127964.1 gsusgguggaCfuUfcucucaauuu 1057 A-127956.7asAfsaUfuGfaGfaGfaagUfcC 1276 L96 faCfcAfcsgsa AD-64004.2 A-127955.4GfsusGfgUfgGfaCfUfUfcUfcU 1058 A-127972.1 asAfsaUfuGfaGfaGfaagUfcC 1277fcAfaUfuUfL96 faccacsgsa AD-64005.2 A-127955.12GfsusGfgUfgGfaCfUfUfcUfcU 1059 A-127980.1 asAfsauuGfagAfgaagUfcCfa 1278fcAfaUfuUfL96 ccacsgsa AD-64006.2 A-127990.1 gsusGfgugGfaCfUfUfcUfcUfc1060 A-127991.1 asAfsaUfuGfaGfaGfaagUfcC 1279 AfaUfuuL96 faCfcacsgsaAD-64007.2 A-127996.4 gsusgguggacUfUfcucucaauuu 1061 A-127995.2asAfsAfUfugaGfaGfaagUfCf 1280 L96 caCfcacsgsa AD-64008.2 A-127955.1GfsusGfgUfgGfaCfUfUfcUfcU 1062 A-127956.1 asAfsaUfuGfaGfaGfaagUfcC 1281fcAfaUfuUfL96 faCfcAfcsgsa AD-64008.2 A-127955.1GfsusGfgUfgGfaCfUfUfcUfcU 1063 A-127956.1 asAfsaUfuGfaGfaGfaagUfcC 1282fcAfaUfuUfL96 faCfcAfcsgsa AD-64008.4 A-127955.15GfsusGfgUfgGfaCfUfUfcUfcU 1064 A-127956.14 asAfsaUfuGfaGfaGfaagUfcC 1283fcAfaUfuUfL96 faCfcAfcsgsa AD-64009.2 A-127965.1gsusgguggacuUfcucucaauuuL 1065 A-127956.8 asAfsaUfuGfaGfaGfaagUfcC 128496 faCfcAfcsgsa AD-64010.2 A-127955.5 GfsusGfgUfgGfaCfUfUfcUfcU 1066A-127973.1 asAfsauuGfagaGfaagUfcCfa 1285 fcAfaUfuUTL96 ccAfcsgsaAD-64011.2 A-127955.13 GfsusGfgUfgGfaCfUfUfcUfcU 1067 A-127981.1asAfsauuGfagagaagUfcCfac 1286 fcAfaUfuUTL96 cacsgsa AD-64012.2A-127990.2 gsusGfgugGfaCfUfUfcUfcUfc 1068 A-127992.1asAfsAfUfuGfaGfaGfaagUfc 1287 AfaUfuuL96 CfaCfcacsgsa AD-64013.2A-127997.1 gsusgguggacdTdTcucucaauuu 1069 A-127998.1asdAsAfuugaGfaGfaagdTdCc 1288 L96 aCfcacsgsa AD-64014.2 A-127957.1Y44GfsusGfgUfgGfaCfUfUfcU 1070 A-127958.1 PasAfsaUfuGfaGfaGfaagUfc 1289fcUfcAfaUfuUfL96 CfaCfcAfcsgsa AD-64015.2 A-127966.1gsusgguggaCfuUfcucuc(Agn) 1071 A-127956.9 asAfsaUfuGfaGfaGfaagUfcC 1290auuuL96 faCfcAfcsgsa AD-64016.2 A-127955.6 GfsusGfgUfgGfaCfUfLfcUfcU1072 A-127974.1 asAfsauuGfaGfaGfaagUfcCf 1291 fcAfaUfuUfL96 accacsgsaAD-64017.2 A-127968.2 gsusgguggacudTcucuc(Agn)a 1073 A-127982.1asdAsauugagagaagdTccacca 1292 uuuL96 csgsa AD-64018.2 A-127990.3gsusGfgugGfaCfUfLfcUfcUfc 1074 A-127993.1 asAfsAfuuGfaGfaGfaagUfCf 1293AfaUfuuL96 caCfcacsgsa AD-64019.2 A-127959.1 gsusggUfgGfaCfUfLfcUfcucA1075 A-127956.2 asAfsaUfuGfaGfaGfaagUfcC 1294 fauuUfL96 faCfcAfcsgsaAD-64020.2 A-127967.1 gsusgguggacuUfcucuc(Agn)a 1076 A-127956.10asAfsaUfuGfaGfaGfaagUfcC 1295 uuuL96 faCfcAfcsgsa AD-64021.2 A-127955.7GfsusGfgUfgGfaCfUfUfcUfcU 1077 A-127975.1 asAfsaUfugaGfaGfaagUfcCf 1296fcAfaUfuUfL96 accAfcsgsa AD-64022.2 A-127968.3 gsusgguggacudTcucuc(Agn)a1078 A-127983.1 PasdAsauugagagaagdTccacc 1297 uuuL96 acsgsa AD-64023.2A-127990.4 gsusGfgugGfaCfUfUfcUfcUfc 1079 A-127994.1asAfsAfUfuGfaGfaGfaagUfC 1298 AfaUfuuL96 fcaCfcacsgsa AD-64024.2A-127960.1 gsusggUfgGfaCfUfUfcUfcucA 1080 A-127956.3asAfsaUfuGfaGfaGfaagUfcC 1299 fauuuL96 faCfcAfcsgsa AD-64025.2A-127968.1 gsusgguggacudTcucuc(Agn)a 1081 A-127956.11asAfsaUfuGfaGfaGfaagUfcC 1300 uuuL96 faCfcAfcsgsa AD-64026.2 A-127955.8GfsusGfgUfgGfaCfUfUfcUfcU 1082 A-127976.1 asAfsaUfugaGfagaagUfcCfa 1301fcAfaUfuUfL96 ccAfcsgsa AD-64027.2 A-127984.1 gsgUfgGfaCfUfUfcUfcUfcAfa1083 A-127985.1 asAfsaUfuGfaGfaGfaagUfcC 1302 UfuUfL96 faCfcsascAD-64028.2 A-127990.5 gsusGfgugGfaCfUfUfcUfcUfc 1084 A-127995.1asAfsAfUfugaGfaGfaagUfCf 1303 AfaUfuuL96 caCfcacsgsa AD-64272.2A-128001.2 GfsusGfcAfcUfuCfGfCfuUfcA 1085 A-128002.2csAfsgAfgGfuGfaAfgcgAfaG 1304 fcCfuCfuGfL96 fuGfcAfcsasc AD-64274.1A-128363.1 GfsusUfgAfcAfaAfAfAfuCfcU 1086 A-128364.1asUfsuGfuGfaGfgAfuuuUfuG 1305 fcAfcAfaUfL96 fuCfaAfcsasa AD-64275.1A-128377.1 UfsgsUfuGfaCfaAfAfAfaUfcC 1087 A-128378.1usUfsgUfgAfgGfaUfuuuUfgU 1306 fuCfaCfaAfL96 fcAfaCfasasg AD-64276.1A-128393.1 GfsgsUfgGfaCfuUfCfUfcUfcA 1088 A-128394.1usAfsaAfaUfuGfaGfagaAfgU 1307 faUfuUfuAfL96 fcCfaCfcsasc AD-64277.1A-128407.1 UfscsUfuUfuGfgAfGfUfgUfgG 1089 A-128408.1usCfsgAfaUfcCfaCfacuCfcA 1308 faUfuCfgAfL96 faAfaGfascsa AD-64277.1A-128407.1 UfscsUfuUfuGfgAfGfUfgUfgG 1090 A-128408.1usCfsgAfaUfcCfaCfacuCfcA 1309 faUfuCfgAfL96 faAfaGfascsa AD-64278.1A-128423.1 AfscsUfgUfuCfaAfGfCfcUfcC 1091 A-128424.1usAfsgCfuUfgGfaGfgcuUfgA 1310 faAfgCfuAfL96 faCfaAfgsasc AD-64279.1A-128435.1 UfscsUfgCfcGfaUfCfCfaUfaC 1092 A-128436.1usCfscGfcAfgUfaUfggaUfcG 1311 fuGfcGfgAfL96 fgCfaGfasgsg AD-64280.1A-128379.1 AfsusGfuGfuCfuGfCfGfgCfgU 1093 A-128380.1usAfsuAfaAfaCfgCfcgcAfgA 1312 fuUfuAfuAfL96 fcAfcAfuscsc AD-64281.1A-128395.1 CfscsCfcGfuCfuGfUfGfcCfuU 1094 A-128396.1usAfsuGfaGfaAfgGfcacAfgA 1313 fcUfcAfuAfL96 fcGfgGfgsasg AD-64282.1A-128409.1 GfscsCfuAfaUfcAfUfCfuCfuU 1095 A-128410.1asUfsgAfaCfaAfgAfgauGfaU 1314 fgUfuCfaUfL96 fuAfgCfgsasg AD-64283.1A-128425.1 UfscsUfaGfaCfuCfGfUfgGfuG 1096 A-128426.1gsAfsaGfuCfcAfcCfacgAfgU 1315 fgAfcUfuCfL96 fcUfaGfascsu AD-64284.1A-128437.1 CfsusGfcCfgAfuCfCfAfuAfcU 1097 A-128438.1usUfscCfgCfaGfuAfuggAfuC 1316 fgCfgGfaAfL96 fgGfcAfgsasg AD-64285.1A-128365.1 UfsusUfuUfcUfuGfUfUfgAfcA 1098 A-128366.1usAfsuUfuUfuGfuCfaacAfaG 1317 faAfaAfuAfL96 faAfaAfascsc AD-64286.1A-128381.1 AfsusCfuUfcUfuGfUfUfgGfuU 1099 A-128382.1usAfsgAfaGfaAfcCfaacAfaG 1318 fcUfuCfuAfL96 faAfgAfusgsa AD-64289.1A-128367.1 GfsusUfuUfuCfuUfGfUfuGfaC 1100 A-128368.1asUfsuUfuUfgUfcAfacaAfgA 1319 faAfaAfaUfL96 faAfaAfcscsc AD-64290.1A-128383.1 CfsusGfcCfuAfaUfCfAfuCfuC 1101 A-128384.1usAfsaCfaAfgAfgAfugaUfuA 1320 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ascsucguggugdGacuu(Cgn)uc 1177 A-127906.20usGfsaGfaGfaAfgUfccaCfcA 1396 ucaL96 fcGfaGfuscsu AD-64711.1 A-129381.1ascsucguggdTgdTacuucdAcuc 1178 A-127906.28 usGfsaGfaGfaAfgUfccaCfcA 1397aL96 fcGfaGfuscsu AD-64712.1 A-127905.22 AfscsUfcGfuGfgUfGfGfaCfuU 1179A-129389.1 usdGsagadGaaguccadCcacga 1398 fcUfcUfcAfL96 guscsu AD-64713.1A-127905.30 AfscsUfcGfuGfgUfGfGfaCfuU 1180 A-129397.1PusgsagadGaagdTccadCcacg 1399 fcUfcUfcAfL96 aguscsu AD-64714.1A-129384.2 ascsucguggdTgdGacuucdAcuc 1181 A-129385.7usdGsagagaagdTccadCcacga 1400 aL96 guscsu AD-64715.1 A-129376.4ascsucguggugdGacuucdAcuca 1182 A-129391.3 usdGsagadGaagdTccadCcacg 1401L96 aguscsu AD-64716.1 A-129374.1 ascsucguggugdGacuucu(Cgn) 1183A-127906.21 usGfsaGfaGfaAfgUfccaCfcA 1402 ucaL96 fcGfaGfuscsu AD-64717.1A-129382.1 ascsucguggdTgdGacuuc(Tgn) 1184 A-127906.29usGfsaGfaGfaAfgUfccaCfcA 1403 cucaL96 fcGfaGfuscsu AD-64718.1A-127905.23 AfscsUfcGfuGfgUfGfGfaCfuU 1185 A-129390.1usdGsagagadAguccadCcacga 1404 fcUfcUfcAfL96 guscsu AD-64719.1 A-127917.5ascsucguggugdGacuuc(Tgn)c 1186 A-129385.2 usdGsagagaagdTccadCcacga 1405ucaL96 guscsu AD-64720.1 A-129381.2 ascsucguggdTgdTacuucdAcuc 1187A-129385.8 usdGsagagaagdTccadCcacga 1406 aL96 guscsu AD-64721.1A-129382.4 ascsucguggdTgdGacuuc(Tgn) 1188 A-129391.4usdGsagadGaagdTccadCcacg 1407 cucaL96 aguscsu AD-64722.1 A-129375.1ascsucguggugdGacuucY34cuc 1189 A-127906.22 usGfsaGfaGfaAfgUfccaCfcA 1408aL96 fcGfaGfuscsu AD-64723.1 A-129383.1 ascsucguggugdGdAcuuc(Tgn) 1190A-127906.30 usGfsaGfaGfaAfgUfccaCfcA 1409 cucaL96 fcGfaGfuscsuAD-64725.1 A-127917.6 ascsucguggugdGacuuc(Tgn)c 1191 A-129398.1PusdGsagagaagdTccadCcacg 1410 ucaL96 aguscsu AD-64726.1 A-129373.3ascsucguggugdGacuu(Cgn)uc 1192 A-129389.2 usdGsagadGaaguccadCcacga 1411ucaL96 guscsu AD-64727.1 A-129384.4 ascsucguggdTgdGacuucdAcuc 1193A-129391.5 usdGsagadGaagdTccadCcacg 1412 aL96 aguscsu AD-64728.1A-129376.1 ascsucguggugdGacuucdAcuca 1194 A-127906.23usGfsaGfaGfaAfgUfccaCfcA 1413 L96 fcGfaGfuscsu AD-64729.1 A-129384.1ascsucguggdTgdGacuucdAcuc 1195 A-127906.31 usGfsaGfaGfaAfgUfccaCfcA 1414aL96 fcGfaGfuscsu AD-64730.1 A-127905.25 AfscsUfcGfuGfgUfGfGfaCfuU 1196A-129392.1 usGsagagaagdTccadCcacgag 1415 fcUfcUfcAfL96 uscsu AD-64731.1A-129399.1 Y34ascsucguggugdGacuuc 1197 A-129385.3usdGsagagaagdTccadCcacga 1416 (Tgn)cucaL96 guscsu AD-64732.1 A-129376.3ascsucguggugdGacuucdAcuca 1198 A-129389.3 usdGsagadGaaguccadCcacga 1417L96 guscsu AD-64733.1 A-129381.4 ascsucguggdTgdTacuucdAcuc 1199A-129391.6 usdGsagadGaagdTccadCcacg 1418 aL96 aguscsu AD-64734.1A-129377.1 ascsucguggugdGacuucdCcuca 1200 A-127906.24usGfsaGfaGfaAfgUfccaCfcA 1419 L96 fcGfaGfuscsu AD-64735.1 A-127905.18AfscsUfcGfuGfgUfGfGfaCfuU 1201 A-129385.1 usdGsagagaagdTccadCcacga 1420fcUfcUfcAfL96 guscsu AD-64736.1 A-127905.26 AfscsUfcGfuGfgUfGfGfaCfuU1202 A-129393.1 usdGsagagaagdTccaCcacgag 1421 fcUfcUfcAfL96 uscsuAD-64737.1 A-129399.2 Y34ascsucguggugdGacuuc 1203 A-129398.2PusdGsagagaagdTccadCcacg 1422 (Tgn)cucaL96 aguscsu AD-64738.1 A-129382.3ascsucguggdTgdGacuuc(Tgn) 1204 A-129389.4 usdGsagadGaaguccadCcacga 1423cucaL96 guscsu AD-64739.1 A-129378.1 ascsucguggugdGacuucdGcuca 1205A-127906.25 usGfsaGfaGfaAfgUfccaCfcA 1424 L96 fcGfaGfuscsu AD-64740.1A-127905.19 AfscsUfcGfuGfgUfGfGfaCfuU 1206 A-129386.1usgsagagaagdTccadCcacgag 1425 fcUfcUfcAfL96 uscsu AD-64741.1 A-127905.27AfscsUfcGfuGfgUfGfGfaCfuU 1207 A-129394.1 usGsagagaagdTccaCcacgagu 1426fcUfcUfcAfL96 scsu AD-64742.1 A-129373.2 ascsucguggugdGacuu(Cgn)uc 1208A-129385.4 usdGsagagaagdTccadCcacga 1427 ucaL96 guscsu AD-64743.1A-129384.3 ascsucguggdTgdGacuucdAcuc 1209 A-129389.5usdGsagadGaaguccadCcacga 1428 aL96 guscsu

TABLE 5 HBV single dose screen using Dual-Glo Luciferase ® Assay DuplexID 10 nM Avg 0.1 nM Avg 10 nM SD 0.1 nM_SD AD-63938.2 0.12 ND 0.01 NDAD-63950.2 0.38 ND 0.04 ND AD-63956.2 0.31 ND 0.02 ND AD-63962.2 0.16 ND0.03 ND AD-63968.2 0.56 ND 0.10 ND AD-63968.2 0.79 ND 0.09 ND AD-63979.20.54 ND 0.02 ND AD-63939.2 0.51 ND 0.01 ND AD-63945.2 0.54 ND 0.08 NDAD-63951.2 0.60 ND 0.03 ND AD-63957.2 0.57 ND 0.02 ND AD-63963.2 0.91 ND0.06 ND AD-63969.2 0.92 ND 0.02 ND AD-63975.2 0.83 ND 0.01 ND AD-63980.20.77 ND 0.01 ND AD-63940.2 0.77 ND 0.06 ND AD-63946.2 0.60 ND 0.10 NDAD-63952.2 0.48 ND 0.04 ND AD-63958.2 0.51 ND 0.01 ND AD-63964.2 0.58 ND0.04 ND AD-63970.2 0.69 ND 0.07 ND AD-63976.2 0.63 ND 0.04 ND AD-63981.20.60 ND 0.04 ND AD-63941.2 0.56 ND 0.09 ND AD-63947.2 0.55 ND 0.08 NDAD-63953.2 0.56 ND 0.06 ND AD-63959.2 0.51 ND 0.03 ND AD-63965.2 0.55 ND0.03 ND AD-63971.2 0.65 ND 0.02 ND AD-63977.2 0.88 ND 0.01 ND AD-63982.20.73 ND 0.07 ND AD-63942.2 0.32 ND 0.09 ND AD-63948.2 0.57 ND 0.09 NDAD-63960.2 0.92 ND 0.05 ND AD-63966.2 0.85 ND 0.06 ND AD-63972.2 0.82 ND0.06 ND AD-63978.2 0.83 ND 0.02 ND AD-63983.2 0.89 ND 0.02 ND AD-63943.20.86 ND 0.04 ND AD-63949.2 0.76 ND 0.02 ND AD-63955.2 0.82 ND 0.02 NDAD-63961.2 0.83 ND 0.07 ND AD-63967.2 0.86 ND 0.03 ND AD-63973.2 0.86 ND0.03 ND AD-63990.2 0.27 ND 0.07 ND AD-63996.2 0.29 ND 0.06 ND AD-64002.20.30 ND 0.11 ND AD-64008.2 0.28 ND 0.05 ND AD-64008.2 0.34 ND 0.07 NDAD-64014.2 0.30 ND 0.03 ND AD-64019.2 0.36 ND 0.04 ND AD-64024.2 0.27 ND0.03 ND AD-63985.2 0.28 ND 0.06 ND AD-63991.2 0.33 ND 0.02 ND AD-63997.20.47 ND 0.07 ND AD-64003.2 0.69 ND 0.06 ND AD-64009.2 0.91 ND 0.03 NDAD-64015.2 0.69 ND 0.09 ND AD-64020.2 0.81 ND 0.06 ND AD-64025.2 0.77 ND0.06 ND AD-63986.2 0.28 ND 0.05 ND AD-63992.2 0.44 ND 0.04 ND AD-64004.20.45 ND 0.04 ND AD-64010.2 0.37 ND 0.05 ND AD-64016.2 0.48 ND 0.05 NDAD-64021.2 0.39 ND 0.03 ND AD-64026.2 0.30 ND 0.02 ND AD-63987.2 0.20 ND0.02 ND AD-63993.2 0.33 ND 0.02 ND AD-63999.2 0.36 ND 0.05 ND AD-64005.20.45 ND 0.11 ND AD-64011.2 0.39 ND 0.08 ND AD-64017.2 0.84 ND 0.06 NDAD-64022.2 0.81 ND 0.03 ND AD-64027.2 0.38 ND 0.05 ND AD-63988.2 0.37 ND0.04 ND AD-63994.2 0.23 ND 0.01 ND AD-64000.2 0.29 ND 0.00 ND AD-64006.20.40 ND 0.04 ND AD-64012.2 0.45 ND 0.17 ND AD-64018.2 0.65 ND 0.07 NDAD-64023.2 0.53 ND 0.07 ND AD-64028.2 0.52 ND 0.07 ND AD-63989.2 0.47 ND0.04 ND AD-63995.2 0.81 ND 0.03 ND AD-64001.2 0.83 ND 0.04 ND AD-64007.20.87 ND 0.04 ND AD-64013.2 0.88 ND 0.03 ND AD-64289.1 0.276 ND 0.009 NDAD-64333.1 0.208 ND 0.015 ND AD-64285.1 0.324 ND 0.034 ND AD-64300.10.225 ND 0.005 ND AD-64345.1 0.102 ND 0.090 ND AD-64292.1 0.288 ND 0.232ND AD-64336.1 0.199 ND 0.056 ND AD-64275.1 0.287 ND 0.185 ND AD-64316.10.297 ND 0.024 ND AD-64274.1 0.209 ND 0.033 ND AD-64315.1 0.199 ND 0.002ND AD-64305.1 0.360 ND 0.035 ND AD-64351.1 0.281 ND 0.014 ND AD-64291.10.725 ND 0.005 ND AD-64335.1 0.478 ND 0.020 ND AD-64283.1 0.917 ND 0.018ND AD-64304.1 0.937 ND 0.050 ND AD-64325.1 0.446 ND 0.223 ND AD-64350.10.934 ND 0.055 ND AD-63968.4 0.748 ND 0.008 ND AD-64331.1 0.294 ND 0.038ND AD-64008.4 0.416 ND 0.028 ND AD-64337.1 0.318 ND 0.049 ND AD-64295.10.415 ND 0.034 ND AD-64276.1 0.453 ND 0.073 ND AD-64317.1 0.203 ND 0.040ND AD-64330.1 0.313 ND 0.030 ND AD-64298.1 0.797 ND 0.007 ND AD-64343.10.667 ND 0.020 ND AD-61547.2 0.637 ND 0.019 ND AD-64347.1 0.418 ND 0.066ND AD-64280.1 0.754 ND 0.092 ND AD-64322.1 0.407 ND 0.013 ND AD-64308.10.720 ND 0.055 ND AD-64354.1 0.315 ND 0.034 ND AD-64303.1 0.815 ND 0.150ND AD-64349.1 0.447 ND 0.030 ND AD-64299.1 0.831 ND 0.007 ND AD-64344.10.404 ND 0.009 ND AD-64309.1 0.856 ND 0.005 ND AD-64355.1 0.498 ND 0.040ND AD-64297.1 0.895 ND 0.024 ND AD-64342.1 0.508 ND 0.006 ND AD-64312.10.590 ND 0.034 ND AD-64358.1 0.425 ND 0.044 ND AD-64341.1 0.223 ND 0.119ND AD-64310.1 0.301 ND 0.064 ND AD-64356.1 0.336 ND 0.024 ND AD-64286.10.611 ND 0.012 ND AD-64328.1 0.317 ND 0.043 ND AD-61522.2 0.447 ND 0.008ND AD-64321.1 0.237 ND 0.009 ND AD-64302.1 0.523 ND 0.020 ND AD-64348.10.208 ND 0.003 ND AD-64352.1 0.343 ND 0.224 ND AD-64352.1 0.567 ND 0.015ND AD-64314.1 0.920 ND 0.044 ND AD-64360.1 0.778 ND 0.029 ND AD-64279.10.882 ND 0.034 ND AD-64320.1 0.589 ND 0.017 ND AD-64284.1 0.696 ND 0.119ND AD-64326.1 0.552 ND 0.009 ND AD-64281.1 0.921 ND 0.019 ND AD-64323.10.715 ND 0.097 ND AD-64311.1 0.815 ND 0.030 ND AD-64357.1 0.549 ND 0.001ND AD-64272.2 0.965 ND 0.024 ND AD-64332.1 0.548 ND 0.013 ND AD-64293.10.837 ND 0.013 ND AD-64338.1 0.597 ND 0.031 ND AD-64290.1 0.489 ND 0.026ND AD-64334.1 0.368 ND 0.003 ND AD-64282.1 0.767 ND 0.009 ND AD-64324.10.726 ND 0.077 ND AD-64278.1 0.951 ND 0.077 ND AD-64319.1 0.895 ND 0.029ND AD-64307.1 0.890 ND 0.065 ND AD-64353.1 0.567 ND 0.500 ND AD-64277.10.416 ND 0.019 ND AD-64277.1 0.839 ND 0.058 ND AD-64318.1 0.613 ND 0.042ND AD-64318.1 0.768 ND 0.042 ND AD-64313.1 0.698 ND 0.062 ND AD-64359.10.441 ND 0.081 ND AD-64294.1 0.563 ND 0.066 ND AD-64339.1 0.486 ND 0.044ND AD-63968.5 0.57 0.72 0.07 0.03 AD-63940.3 0.81 0.83 0.11 0.03AD-64710.1 0.79 0.85 0.12 0.04 AD-64716.1 0.73 0.85 0.08 0.01 AD-64722.10.67 0.80 0.06 0.02 AD-64728.1 0.74 0.87 0.06 0.05 AD-64734.1 0.78 0.830.08 0.05 AD-64739.1 0.73 0.85 0.07 0.02 AD-64700.1 0.54 0.75 0.13 0.02AD-64705.1 0.67 0.79 0.15 0.04 AD-64711.1 0.57 0.83 0.13 0.04 AD-64717.10.72 0.83 0.13 0.02 AD-64723.1 0.83 0.87 0.12 0.01 AD-64729.1 0.74 0.870.08 0.07 AD-64735.1 0.73 0.89 0.05 0.04 AD-64740.1 0.89 0.88 0.05 0.07AD-64701.1 0.88 0.84 0.07 0.05 AD-64706.1 0.71 0.88 0.12 0.05 AD-64712.10.81 0.86 0.13 0.07 AD-64718.1 0.84 0.89 0.16 0.01 AD-64730.1 0.88 0.890.02 0.04 AD-64736.1 0.80 0.88 0.10 0.05 AD-64741.1 0.85 0.83 0.06 0.05AD-64702.1 0.87 0.93 0.02 0.06 AD-64707.1 0.95 0.88 0.05 0.08 AD-64713.10.90 0.85 0.08 0.03 AD-64719.1 0.80 0.89 0.09 0.09 AD-64725.1 0.70 0.840.09 0.03 AD-64731.1 0.82 0.87 0.04 0.08 AD-64737.1 0.76 0.84 0.09 0.08AD-64742.1 0.76 0.85 0.09 0.03 AD-64703.1 0.79 0.88 0.05 0.02 AD-64708.10.83 0.82 0.08 0.06 AD-64714.1 0.75 0.85 0.12 0.03 AD-64720.1 0.61 0.810.17 0.04 AD-64726.1 0.75 0.83 0.07 0.02 AD-64732.1 0.86 0.84 0.14 0.10AD-64738.1 0.80 0.90 0.04 0.02 AD-64743.1 0.75 0.85 0.12 0.04 AD-64704.10.67 0.78 0.16 0.02 AD-64709.1 0.83 0.86 0.16 0.03 AD-64715.1 0.87 0.880.09 0.04 AD-64721.1 0.77 0.82 0.12 0.06 AD-64727.1 0.75 0.85 0.14 0.02AD-64733.1 0.67 0.81 0.14 0.03

Example 3. Synthesis and In Vitro Screening of Additional iRNA Duplexes

Additional iRNA molecules targeting the HBV genome were synthesized asdescribed above. A detailed list of the additional modified HBV senseand antisense strand sequences is shown in Table 6.

A single dose screen of these duplexes was performed by transfecting theduplexes into HepG2.215 and Hep3B cells and measuring Firefly(transfection control) and Rinella (fused to HBV target sequence)luciferase, as described above. The results of the assays in HepG2.2.15cells are shown in Table 7 and the results of the assays in Hep3B cellsare provided in Table 8.

TABLE 6 Modified Sense and Antisense Strand Sequences of HBV dsRNAs SEQSEQ ID ID DuplexID Sense Sequence (5′ to 3′) NO:Antisense Sequence (5′ to 3′) NO: AD-65369uscsguGfgUfGfGfacuuCfUfcucaL96 1429 PusGfsagaGfaAfGfuccaCfcAfcgasusu1458 AD-65381 uscsguGfgUfGfGfacuucucucaL96 1430PusGfsagaGfaAfGfuccaCfcAfcgasusu 1459 AD-63962Y44uscsGfuGfgUfgGfaCfuUfcUfcUfcAfY44 1431PusGfsaGfaGfaAfgUfcCfaCfcAfcGfasusu 1460 AD-63938Y44ACUCGUGGUGGACUUCUCUCA 1432 UGAGAGAAGUCCACCACGAGUCU 1461 AD-65561uscsguGfgUfGfGfacuuCfUfcucaL96 1433 UfsGfsagaGfaAfGfuccaCfcAfcgasusu1462 AD-65566 uscsguGfgUfGfGfacuucucucaL96 1434UfsGfsagaGfaAfGfuccaCfcAfcgasusu 1463 AD-63944Y44ucGuGGuGGAcuucucucAusuY44 1435 UfGfagAfgAfAfGUfccaCfCAfcgAusu 1464AD-63968 AfscsUfcGfuGfgUfGfGfaCfuUfcUfcUfcAfL96 1436usGfsaGfaGfaAfgUfccaCfcAfcGfaGfuscsu 1465 AD-65406uscsguGfgUfGfGfacuuCfUfcucaL96 1437 usGfsagaGfaAfGfuccaCfcAfcgasusu 1466AD-65396 ascsucguGfgUfGfGfacuucucucaL96 1438usGfsagaGfaaguccaCfcAfcgagususu 1467 AD-65427gsusgcacUfuCfGfCfuucaccucuaL96 1439 PusAfsgagGfugaagcgAfaGfugcacsusu1468 AD-65573 gsusgcacUfuCfGfCfuucaCfCfucuaL96 1440UfsAfsgagGfuGfAfagcgAfaGfugcacsasc 1469 AD-65432gscsacUfucGfCfuucacCfucuaL96 1441 PusAfsgagGfuGfAfagcgAfaGfugcsasc 1470AD-64332 GfsusGfcAfcUfuCfGfCfuUfcAfcCfuCfuGfL96 1442PcsAfsgAfgGfuGfaAfgcgAfaGfuGfcAfcsasc 1471 AD-64322AfsusGfuGfuCfuGfCfGfgCfgUfuUfuAfuAfL96 1443PusAfsuAfaAfaCfgCfcgcAfgAfcAfcAfuscsc 1472 AD-64272GfsusGfcAfcUfuCfGfCfuUfcAfcCfuCfuGfL96 1444csAfsgAfgGfuGfaAfgcgAfaGfuGfcAfcsasc 1473 AD-65583gscsacuucgdCuucac(Cgn)ucuaL96 1445 usdAsgagdGugaagcgdAagugcsusu 1474AD-63994 gsgsUfgGfaCfUfUfcUfcUfcAfaUfuUTL96 1446PasAfsaUfuGfaGfaGfaagUfcCfaCfcsasc 1475 AD-65370csgsugguGfgAfCfUfucucUfCfaauuL96 1447 asAfsuugAfgAfGfaaguCfcAfccagcsasg1476 AD-65265 gsusggugGfaCfUfUfcUfcucaauuuL96 1448asAfsaUfugagaGfaagUfcCfaccAfcsgsa 1477 AD-65407csgsugguGfgAfCfUfucucUfCfaauuL96 1449 asAfsuugAfgAfgAfaguCfcAfccagcsasg1478 AD-64027 gsgsUfgGfaCfUfUfcUfcUfcAfaUfuUTL96 1450asAfsaUfuGfaGfaGfaagUfcCfaCfcsasc 1479 AD-65266gsusggugGfaCfUfUfcucuCfaauuuL96 1451 asAfsaUfugagaGfaagUfcCfaccAfcsgsa1480 AD-65389 usgsgudGgucdTucucuaaauuL96 1452asdAsuugagagdAagudCcaccasusu 1481 AD-64008GfsusGfgUfgGfaCfUfUfcUfcUfcAfaUfuUTL96 1453asAfsaUfuGfaGfaGfaagUfcCfaCfcAfcsgsa 1482 AD-65377csgsuggudGgucdTucucuaaauuL96 1454 asdAsuugagagdAagudCcaccagcsusu 1483AD-65409 gsgsuggaCfuUTCfUfcucaAfUfuuuaL96 1455PusAfsaaaUfuGfAfgagaAfgUfccaccsasc 1484 AD-65403gsgsuggaCfuUTCfUfcucaAfUfuuuaL96 1456 usAfsaaaUfuGfAfgagaAfgUfccaccsasc1485 AD-65385 usgsgacuacdTcucaaauuuaL96 1457usdAsaaauugadGagadAguccasusu 1486

TABLE 7 HBV single dose screen In HepG2.2.15 cells using Dual-GloLuciferase ® Assay Duplex ID PORF-1_A PORF-1_B SORF-2_A SORF-2_BAD-65369 0.1875 0.042 0.0446 0.3018 AD-65381 0.086 0.249 0.1008 0.553AD-63962 0.4838 0.3475 0.2237 0.5258 AD-63938 0.3587 2.1213 0.05011.1434 AD-65561 0.1076 0.3801 0.0718 0.6897 AD-65566 0.4127 0.3211 0.18511.1161 AD-63944 0.9489 0.7098 0.393 0.2771 AD-63968 NoIC50 NoIC501.8788 NoIC50 AD-65406 3.3749 18.8396 3.8204 2.2662 AD-65396 NoIC506.8758 3.7382 4.2157 AD-65427 0.0089 0.0181 0.0066 0.015 AD-65573 0.01740.0332 0.0029 0.0227 AD-65432 0.0211 0.0593 0.0112 0.0366 AD-643320.0268 0.0329 0.0624 0.0217 AD-64322 0.0963 0.1077 0.0992 0.0963AD-64272 0.0773 0.1199 0.0763 0.093 AD-65583 0.1624 0.2228 0.1568 0.1496AD-63994 0.7019 0.1467 0.0832 0.0385 AD-65370 0.2404 0.7916 0.39520.1964 AD-65265 0.2255 0.5008 0.2893 0.318 AD-65407 0.9533 0.261 0.42540.1121 AD-64027 0.7692 0.5887 0.5208 0.5697 AD-65266 3.4109 0.50550.8532 0.3658 AD-65389 0.9172 0.6514 0.4915 0.2872 AD-64008 1.27380.7865 1.9519 0.808 AD-65377 0.6052 1.6 24.9403 0.6065 AD-65409 1.83041.6479 0.104 0.0557 AD-65403 12.1516 0.667 1.006 0.233 AD-65385 NoIC50NoIC50 NoIC50 NoIC50

TABLE 8 HBV single dose screen In Hep3B cells using Dual-GloLuciferase  ® Assay Duplex ID PORF-1_A PORF-1_B AD-65369 0.0982 0.0508AD-65381 0.2392 0.1097 AD-63962 0.0769 0.0706 AD-63938 0.039 0.0111AD-65561 0.6316 0.6931 AD-65566 0.2747 0.5331 AD-63944 0.1317 0.0566AD-63968 0.4374 0.8811 AD-65406 1.4961 1.2573 AD-65396 1.9971 0.9952AD-65427 0.0234 0.006 AD-65573 0.0346 0.0334 AD-65432 0.0352 0.2664AD-64332 0.0221 0.4541 AD-64322 0.1743 0.1616 AD-64272 0.1885 0.6699AD-65583 0.1241 8.1611 AD-63994 3.3623 5.2897 AD-65370 0.2281 NoIC50AD-65265 NoIC50 7.3426 AD-65407 0.1404 1.3833 AD-64027 27.1417 1.1832AD-65266 NoIC50 NoIC50 AD-65389 NoIC50 NoIC50 AD-64008 NoIC50 NoIC50AD-65377 NoIC50 NoIC50 AD-65409 1.8065 3.436 AD-65403 0.5113 18.0359AD-65385 NoIC50 NoIC50

A subset of these duplexes were also assayed for in vitro stabilityusing two assays, a tritosome stability assay and a cytosol stabilityassay.

For the tritosome stability assays, rat liver tritosomes (Xenotechcustom product PR14044) were thawed to room temperature and diluted to0.5 units/mL Acid Phosphatase in 20 mM Sodium Citrate pH 5.0 Buffer.Twenty-four hour samples were prepared by mixing 100 μL of 0.5 units/mLAcid Phosphatase Tritosomes with 25 μL of 0.4 mg/mL siRNA sample in amicrocentrifuge tube and incubating for twenty-four hours in aneppendorf Thermomixer set to 37° C. and 300 rpm. After twenty-four hoursof incubation 300 μL of Phenomenex Lysis Loading Buffer (Cat. #ALO-8498) and 12.5 μL of a 0.4 mg/mL internal standard siRNA were addedto each sample. Time 0 hour samples were prepared by by mixing 100 μL of0.5 units/mL Acid Phosphatase Tritosomes with 25 μL of 0.4 mg/mL siRNAsample, 300 μL of Phenomenex Lysis Loading Buffer, and 12.5 μL of a 0.4mg/mL internal standard siRNA. siRNA was extracted from twenty-four hoursamples and 0 hour samples using a Phenomenex Clarity OTX Starter Kit(Cat. # KSO-8494). After the samples were extracted they weretransferred to a microcentrifuge tube and dried down using a LabconcoCentriVap Concentrator (Cat. #7810010). The samples were thenresuspended with 500 μL of nuclease free water. Fifty μL of each samplewas run on an Agilent Technologies 1260 Infinity Binary LC with AgilentTechnologies 6130 Quadrupole LC/MS. The Quaternary pump method was runfor 12.20 minutes at 0.400 mL/min with the following timetable:

Time Function Parameter 0.20 5% Buffer A(16 mM TEA 200 mM HFIP), 95%Buffer B (100% Methanol) 2.50 5% Buffer A(16 mM TEA 200 mM HFIP), 95%Buffer B (100% Methanol) 3.00 100% Buffer A(16 mM TEA 200 mM HFIP)The Binary Pump method was run for 12.20 min at 0.700 mL/min with thefollowing timetable:

Time Function Parameter 0.00 100% Buffer A(16 mM TEA 200 mM HFIP) 0.40100% Buffer A(16 mM TEA 200 mM HFIP) 10.00 60% Buffer A(16 mM TEA 200 mMHFIP), 40% Buffer B (100% ACN) 10.10 100% Buffer A(16 mM TEA 200 mMHFIP) 12.20 100% Buffer A(16 mM TEA 200 mM HFIP)Both the left and right column was set at 75.00° C. The UV signal wasmeasured at 260 nm wavelength. The percent remaining of each strand wascalculated using the following equation:

% Strand remaining=100*(Peak Areas_(Strand 24 h)/PeakArea_(Strand 0 h)*(Peak Area_(Standard 24 h)/Peak Area_(Standard 0 h))).

For the cytosol stability assay, female rat liver cytosol (Xenotech Cat.# R1500.C) were thawed to room temperature and diluted to 1 mg/mL in 50mM Tris buffer: HCl pH 7.4, 5 mM MgCl2. 24 hour samples were prepared bymixing 100 uL of 1 mg/mL Cytosol with 25 uL of 0.4 mg/mL siRNA sample ina microcentrifuge tube and incubating for 24 hours in an eppendorfThermomixer set to 37° C. and 300 rpm. After 24 hours of incubation 300uL of Phenomenex Lysis Loading Buffer (Cat. # ALO-8498) and 12.5 uL of a0.4 mg/mL internal standard siRNA were added to each sample. 0 hoursamples were prepared by by mixing 100 uL of 1 mg/mL Cytosol with 25 uLof 0.4 mg/mL siRNA sample, 300 uL of Phenomenex Lysis Loading Buffer,and 12.5 uL of a 0.4 mg/mL internal standard siRNA. siRNA was extractedfrom 24 hour samples and 0 hour samples using a Phenomenex Clarity OTXStarter Kit (Cat. # KSO-8494). After the samples were extracted theywere transferred to a microcentrifuge tube and dried down using aLabconco CentriVap Concentrator (Cat. #7810010). The samples were thenresuspended with 500 uL of nuclease free water. 50 uL of each sample wasrun on an Agilent Technologies 1260 Infinity Binary LC with AgilentTechnologies 6130 Quadrupole LC/MS. The Quaternary pump method was runfor 12.20 minutes at 0.400 mL/min with the following timetable:

Time Function Parameter 0.20 5% Buffer A(16 mM TEA 200 mM HFIP), 95%Buffer B (100% Methanol) 2.50 5% Buffer A(16 mM TEA 200 mM HFIP), 95%Buffer B (100% Methanol) 3.00 100% Buffer A(16 mM TEA 200 mM HFIP)The Binary Pump method was run for 12.20 min at 0.700 mL/min with thefollowing timetable:

Time FunctionParameter

Time Function Parameter 0.00 100% Buffer A(16 mM TEA 200 mM HFIP) 0.40100% Buffer A(16 mM TEA 200 mM HFIP) 10.00 60% Buffer A(16 mM TEA 200 mMHFIP), 40% Buffer B (100% ACN) 10.10 100% Buffer A(16 mM TEA 200 mMHFIP) 12.20 100% Buffer A(16 mM TEA 200 mM HFIP)Both the left and right column was set at 75.00° C. The UV signal wasmeasured at 260 nm wavelength. The percent remaining of each strand wascalculated using the following equation:

% Strand remaining=100*(Peak Area_(Strand 24 h)/PeakArea_(Strand 0 h)*(Peak Area_(Standard 24 h)/Peak Area_(Standard 0 h))).

The results of the twenty-four hour tritosome stability assays areprovided in Table 9 and the results of the twenty-four hour cytosolstability assays are provided in Table 10.

TABLE 9 Twenty-four hour tritosome stability assays. % AntisenseRemaining % Sense Remaining DuplexID AD-65369 87.59 72.43 AD-65381AD-63962 AD-63938 AD-65561 67.59 82.48 AD-65566 AD-63944 30.52 34.98AD-63968 AD-65406 AD-65396 115.17 79.61 AD-65427 43.00 76.84 AD-65573AD-65432 AD-64332 AD-64322 129.69 128.59 AD-64272 AD-65583 AD-63994AD-65370 AD-65265 100.30 119.85 AD-65407 AD-64027 AD-65266 AD-6538994.06 110.90 AD-64008 98.63 127.48 AD-65377 105.06 119.88 AD-65409117.55 104.30 AD-65403 AD-65385

TABLE 10 Twenty-four hour cytosol stability assays. % AntisenseRemaining % Sense Remaining DuplexID AD-65369 67.78 22.42 AD-65381AD-63962 AD-63938 AD-65561 55.89 15.26 AD-65566 AD-63944 88.39 46.94AD-63968 AD-65406 AD-65396 89.50 66.35 AD-65427 69.01 41.47 AD-65573AD-65432 AD-64332 AD-64322 96.77 78.00 AD-64272 AD-65583 AD-63994AD-65370 AD-65265 64.46 24.10 AD-65407 AD-64027 AD-65266 AD-65389 35.3926.39 AD-64008 79.98 66.50 AD-65377 86.24 74.25 AD-65409 60.45 62.41AD-65403 AD-65385

Example 4. Synthesis and In Vitro Screening of iRNA Duplexes TargetingHDV

The selection of iRNA designs targeting HDV was driven by two primaryfactors: a) potency and b), the desire to employ iRNA with near-perfectmatches with greater than 90% fractional coverage of the large number ofpublic HDV sequences of all known clades (I-VIII or 1-8). However, dueto the low level of conservation between members of the various HDVclades and the small genome, selection of sequences that effectivelytarget multiple HDV clades was difficult. Exemplary sequences for thevarious HDV clades 1-8 are provided in SEQ ID NOs: 29, 31, 33, 35, 37,39, 41, and 43. Their reverse complements are provided in SEQ ID NOs:30, 32, 34, 36, 38, 40, 42, and 44.

iRNA molecules targeting the HDV genome were synthesized as describedabove. A detailed list of the unmodified HDV sense and antisense strandsequences is shown in Table 11 and a detailed list of the modified HDVsense and antisense strand sequences is shown in Table 12. The resultsof a single dose screen of these agents using the Dual-Glo Luciferase®Assay described above are shown in Table 13.

TABLE 11 Unmodified Sense and Antisense Strand Sequences of HDV dsRNAsSEQ ID NO Duplex Sense Antisense Sense/ Name Strand Strand CladeSense Seq Antisense Seq Antisense AD-45013.1 A-94349.1 A-94350.1 HDV1ACGAAGGAAGGCCCUCGAGdTdT CUCGAGGGCCUUCCUUCGUdTdT  45/46 AD-45019.1A-94351.1 A-94352.1 HDV1 CGAAGGAAGGCCCUCGAGAdTdT UCUCGAGGGCCUUCCUUCGdTdT 47/48 AD-45025.1 A-94353.1 A-94354.1 HDV1 GAAGGAAGGCCCUCGAGAAdTdTUUCUCGAGGGCCUUCCUUCdTdT  49/50 AD-45036.1 A-94357.1 A-94358.1 HDV1AGGAAGGCCCUCGAGAACAdTdT UGUUCUCGAGGGCCUUCCUdTdT  51/52 AD-45041.1A-94359.1 A-94360.1 HDV1 GGAAGGCCCUCGAGAACAAdTdT UUGUUCUCGAGGGCCUUCCdTdT 53/54 AD-45046.1 A-94361.1 A-94362.1 HDV1 GAAGGCCCUCGAGAACAAGdTdTCUUGUUCUCGAGGGCCUUCdTdT  55/56 AD-45051.1 A-94363.1 A-94364.1 HDV1GGGGUGUGAACCCCCUCGAdTdT UCGAGGGGGUUCACACCCCdTdT  57/58 AD-45014.1A-94365.1 A-94366.1 HDV1 GGGUGUGAACCCCCUCGAAdTdT UUCGAGGGGGUUCACACCCdTdT 59/60 AD-45020.1 A-94367.1 A-94368.1 HDV1 GGUGUGAACCCCCUCGAAGdTdTCUUCGAGGGGGUUCACACCdTdT  61/62 AD-45026.1 A-94369.1 A-94370.1 HDV1UGUGAACCCCCUCGAAGGUdTdT ACCUUCGAGGGGGUUCACAdTdT  63/64 AD-45032.1A-94371.1 A-94372.1 HDV2 AAAAUCCCUGGCUGGGGAAdTdT UUCCCCAGCCAGGGAUUUUdTdT 65/66 AD-45042.1 A-94375.1 A-94376.1 HDV2 AAUCCCUGGCUGGGGAACAdTdTUGUUCCCCAGCCAGGGAUUdTdT  67/68 AD-45047.1 A-94377.1 A-94378.1 HDV2AUCCCUGGCUGGGGAACAUdTdT AUGUUCCCCAGCCAGGGAUdTdT  69/70 AD-45015.1A-94381.1 A-94382.1 HDV2 AAGAGCGGGUUCACCGACAdTdT UGUCGGUGAACCCGCUCUUdTdT 71/72 AD-45021.1 A-94383.1 A-94384.1 HDV2 AGAGCGGGUUCACCGACAAdTdTUUGUCGGUGAACCCGCUCUdTdT  73/74 AD-45027.1 A-94385.1 A-94386.1 HDV2GAGCGGGUUCACCGACAAGdTdT CUUGUCGGUGAACCCGCUCdTdT  75/76 AD-45033.1A-94387.1 A-94388.1 HDV2 GCGGGUUCACCGACAAGGAdTdT UCCUUGUCGGUGAACCCGCdTdT 77/78 AD-45038.1 A-94389.1 A-94390.1 HDV2 CGGGUUCACCGACAAGGAGdTdTCUCCUUGUCGGUGAACCCGdTdT  79/80 AD-45043.1 A-94391.1 A-94392.1 HDV2GGGUUCACCGACAAGGAGAdTdT UCUCCUUGUCGGUGAACCCdTdT  81/82 AD-45048.1A-94393.1 A-94394.1 HDV2 GGUUCACCGACAAGGAGAGdTdT CUCUCCUUGUCGGUGAACCdTdT 83/84 AD-45053.1 A-94395.1 A-94396.1 HDV2 GGGAGGGACUGGACAUCAGdTdTCUGAUGUCCAGUCCCUCCCdTdT  85/86 AD-45016.1 A-94397.1 A-94398.1 HDV3GGGUAGAGGAAAGGAAGAAdTdT UUCUUCCUUUCCUCUACCCdTdT  87/88 AD-45022.1A-94399.1 A-94400.1 HDV3 GAGGCGGGACCACAGAAGAdTdT UCUUCUGUGGUCCCGCCUCdTdT 89/90 AD-45028.1 A-94401.1 A-94402.1 HDV3 ACAGAAGAAGGAAGGCCCUdTdTAGGGCCUUCCUUCUUCUGUdTdT  91/92 AD-45034.1 A-94403.1 A-94404.1 HDV3GAAGAAGAGGAACUCCGGAdTdT UCCGGAGUUCCUCUUCUUCdTdT  93/94 AD-45039.1A-94405.1 A-94406.1 HDV3 CAGGGAUGACGACGAAAGAdTdT UCUUUCGUCGUCAUCCCUGdTdT 95/96 AD-45044.1 A-94407.1 A-94408.1 HDV3 GGGAUGACGACGAAAGAGAdTdTUCUCUUUCGUCGUCAUCCCdTdT  97/98 AD-45049.1 A-94409.1 A-94410.1 HDV3GGAUGACGACGAAAGAGAAdTdT UUCUCUUUCGUCGUCAUCCdTdT  99/100 AD-45054.1A-94411.1 A-94412.1 HDV3 CCUGGGGGUGUGAACCCAAdTdT UUGGGUUCACACCCCCAGGdTdT101/102 AD-45017.1 A-94413.1 A-94414.1 HDV3 CUGGGGGUGUGAACCCAAUdTdTAUUGGGUUCACACCCCCAGdTdT 103/104 AD-45023.1 A-94415.1 A-94416.1 HDV3CCUUUUCCCGAACGGGAGAdTdT UCUCCCGUUCGGGAAAAGGdTdT 105/106 AD-45035.1A-94419.1 A-94420.1 HDV3 GAACGGGAGAGGGGAUCGAdTdT UCGAUCCCCUCUCCCGUUCdTdT107/108 AD-45040.1 A-94421.1 A-94422.1 HDV3 ACGGGAGAGGGGAUCGACAdTdTUGUCGAUCCCCUCUCCCGUdTdT 109/110 AD-45050.1 A-94425.1 A-94426.1 HDV3AGAGGGGAUCGACAUCCGAdTdT UCGGAUGUCGAUCCCCUCUdTdT 111/112 AD-45055.1A-94427.1 A-94428.1 HDV3 GGGGAUCGACAUCCGAGGAdTdT UCCUCGGAUGUCGAUCCCCdTdT113/114 AD-45018.1 A-94429.1 A-94430.1 HDV3 GGGAUCGACAUCCGAGGAAdTdTUUCCUCGGAUGUCGAUCCCdTdT 115/116 AD-45024.1 A-94431.1 A-94432.1 HDV3ACAUCCGAGGAACCCAGCAdTdT UGCUGGGUUCCUCGGAUGUdTdT 117/118 AD-45030.1A-94433.1 A-94434.1 HDV3 GGAACCCAGCAGUUCCCAUdTdT AUGGGAACUGCUGGGUUCCdTdT119/120

TABLE 12 Modified Sense and Antisense Strand Sequences of HDV dsRNAsAnti- SEQ SEQ Duplex Sense sense Sense Sequence ID Antisense Sequence IDName Strand Strand Clade (5′ to 3′) NO: (5′ to 3′) NO: AD- A- A- HDV1AcGAAGGAAGGcccucGAGdTsdT 1487 CUCGAGGGCCUUCCUUCGUdTsdT 1525 45013.194349.1 94350.1 AD- A- A- HDV1 cGAAGGAAGGcccucGAGAdTsdT 1488UCUCGAGGGCCUUCCUUCGdTsdT 1526 45019.1 94351.1 94352.1 AD- A- A- HDV1GAAGGAAGGcccucGAGAAdTsdT 1489 UUCUCGAGGGCCUUCCUUCdTsdT 1527 45025.194353.1 94354.1 AD- A- A- HDV1 AGGAAGGcccucGAGAAcAdTsdT 1490UGUUCUCGAGGGCCUUCCUdTsdT 1528 45036.1 94357.1 94358.1 AD- A- A- HDV1GGAAGGcccucGAGAAcAAdTsdT 1491 UUGUUCUCGAGGGCCUUCCdTsdT 1529 45041.194359.1 94360.1 AD- A- A- HDV1 GAAGGcccucGAGAAcAAGdTsdT 1492CUUGUUCUCGAGGGCCUUCdTsdT 1530 45046.1 94361.1 94362.1 AD- A- A- HDV1GGGGuGuGAAcccccucGAdTsdT 1493 UCGAGGGGGUUcAcACCCCdTsdT 1531 45051.194363.1 94364.1 AD- A- A- HDV1 GGGuGuGAAcccccucGAAdTsdT 1494UUCGAGGGGGUUcAcACCCdTsdT 1532 45014.1 94365.1 94366.1 AD- A- A- HDV1GGuGuGAAcccccucGAAGdTsdT 1495 CUUCGAGGGGGUUcAcACCdTsdT 1533 45020.194367.1 94368.1 AD- A- A- HDV1 uGuGAAcccccucGAAGGudTsdT 1496ACCUUCGAGGGGGUUcAcAdTsdT 1534 45026.1 94369.1 94370.1 AD- A- A- HDV2AAAAucccuGGcuGGGGAAdTsdT 1497 UUCCCcAGCcAGGGAUUUUdTsdT 1535 45032.194371.1 94372.1 AD- A- A- HDV2 AAucccuGGcuGGGGAAcAdTsdT 1498UGUUCCCcAGCcAGGGAUUdTsdT 1536 45042.1 94375.1 94376.1 AD- A- A- HDV2AucccuGGcuGGGGAAcAudTsdT 1499 AUGUUCCCcAGCcAGGGAUdTsdT 1537 45047.194377.1 94378.1 AD- A- A- HDV2 AAGAGcGGGuucAccGAcAdTsdT 1500UGUCGGUGAACCCGCUCUUdTsdT 1538 45015.1 94381.1 94382.1 AD- A- A- HDV2AGAGcGGGuucAccGAcAAdTsdT 1501 UUGUCGGUGAACCCGCUCUdTsdT 1539 45021.194383.1 94384.1 AD- A- A- HDV2 GAGcGGGuucAccGAcAAGdTsdT 1502CUUGUCGGUGAACCCGCUCdTsdT 1540 45027.1 94385.1 94386.1 AD- A- A- HDV2GcGGGuucAccGAcAAGGAdTsdT 1503 UCCUUGUCGGUGAACCCGCdTsdT 1541 45033.194387.1 94388.1 AD- A- A- HDV2 cGGGuucAccGAcAAGGAGdTsdT 1504CUCCUUGUCGGUGAACCCGdTsdT 1542 45038.1 94389.1 94390.1 AD- A- A- HDV2GGGuucAccGAcAAGGAGAdTsdT 1505 UCUCCUUGUCGGUGAACCCdTsdT 1543 45043.194391.1 94392.1 AD- A- A- HDV2 GGuucAccGAcAAGGAGAGdTsdT 1506CUCUCCUUGUCGGUGAACCdTsdT 1544 45048.1 94393.1 94394.1 AD- A- A- HDV2GGGAGGGAcuGGAcAucAGdTsdT 1507 CUGAUGUCcAGUCCCUCCCdTsdT 1545 45053.194395.1 94396.1 AD- A- A- HDV3 GGGuAGAGGAAAGGAAGAAdTsdT 1508UUCUUCCUUUCCUCuACCCdTsdT 1546 45016.1 94397.1 94398.1 AD- A- A- HDV3GAGGcGGGAccAcAGAAGAdTsdT 1509 UCUUCUGUGGUCCCGCCUCdTsdT 1547 45022.194399.1 94400.1 AD- A- A- HDV3 AcAGAAGAAGGAAGGcccudTsdT 1510AGGGCCUUCCUUCUUCUGUdTsdT 1548 45028.1 94401.1 94402.1 AD- A- A- HDV3GAAGAAGAGGAAcuccGGAdTsdT 1511 UCCGGAGUUCCUCUUCUUCdTsdT 1549 45034.194403.1 94404.1 AD- A- A- HDV3 cAGGGAuGAcGAcGAAAGAdTsdT 1512UCUUUCGUCGUcAUCCCUGdTsdT 1550 45039.1 94405.1 94406.1 AD- A- A- HDV3GGGAuGAcGAcGAAAGAGAdTsdT 1513 UCUCUUUCGUCGUcAUCCCdTsdT 1551 45044.194407.1 94408.1 AD- A- A- HDV3 GGAuGAcGAcGAAAGAGAAdTsdT 1514UUCUCUUUCGUCGUcAUCCdTsdT 1552 45049.1 94409.1 94410.1 AD- A- A- HDV3ccuGGGGGuGuGAAcccAAdTsdT 1515 UUGGGUUcAcACCCCcAGGdTsdT 1553 45054.194411.1 94412.1 AD- A- A- HDV3 cuGGGGGuGuGAAcccAAudTsdT 1516AUUGGGUUcAcACCCCcAGdTsdT 1554 45017.1 94413.1 94414.1 AD- A- A- HDV3ccuuuucccGAAcGGGAGAdTsdT 1517 UCUCCCGUUCGGGAAAAGGdTsdT 1555 45023.194415.1 94416.1 AD- A- A- HDV3 GAAcGGGAGAGGGGAucGAdTsdT 1518UCGAUCCCCUCUCCCGUUCdTsdT 1556 45035.1 94419.1 94420.1 AD- A- A- HDV3AcGGGAGAGGGGAucGAcAdTsdT 1519 UGUCGAUCCCCUCUCCCGUdTsdT 1557 45040.194421.1 94422.1 AD- A- A- HDV3 AGAGGGGAucGAcAuccGAdTsdT 1520UCGGAUGUCGAUCCCCUCUdTsdT 1558 45050.1 94425.1 94426.1 AD- A- A- HDV3GGGGAucGAcAuccGAGGAdTsdT 1521 UCCUCGGAUGUCGAUCCCCdTsdT 1559 45055.194427.1 94428.1 AD- A- A- HDV3 GGGAucGAcAuccGAGGAAdTsdT 1522UUCCUCGGAUGUCGAUCCCdTsdT 1560 45018.1 94429.1 94430.1 AD- A- A- HDV3AcAuccGAGGAAcccAGcAdTsdT 1523 UGCUGGGUUCCUCGGAUGUdTsdT 1561 45024.194431.1 94432.1 AD- A- A- HDV3 GGAAcccAGcAGuucccAudTsdT 1524AUGGGAACUGCUGGGUUCCdTsdT 1562 45030.1 94433.1 94434.1

TABLE 13 HDV single dose screen using Dual-Glo Luciferase ® Assay 10 nM0.1 nM Duplex mean mean Sense Antisense Name KD KD Strand Strand CladeAD-45013.1 0.564 0.885 A-94349.1 A-94350.1 HDV1 AD-45019.1 0.238 0.749A-94351.1 A-94352.1 HDV1 AD-45025.1 0.203 0.747 A-94353.1 A-94354.1 HDV1AD-45036.1 0.557 0.865 A-94357.1 A-94358.1 HDV1 AD-45041.1 0.710 0.865A-94359.1 A-94360.1 HDV1 AD-45046.1 0.849 0.937 A-94361.1 A-94362.1 HDV1AD-45051.1 0.863 0.909 A-94363.1 A-94364.1 HDV1 AD-45014.1 0.556 0.883A-94365.1 A-94366.1 HDV1 AD-45020.1 0.860 0.949 A-94367.1 A-94368.1 HDV1AD-45026.1 0.793 0.847 A-94369.1 A-94370.1 HDV1 AD-45032.1 0.569 0.878A-94371.1 A-94372.1 HDV2 AD-45042.1 0.913 0.928 A-94375.1 A-94376.1 HDV2AD-45047.1 0.593 0.858 A-94377.1 A-94378.1 HDV2 AD-45015.1 0.615 0.948A-94381.1 A-94382.1 HDV2 AD-45021.1 0.856 0.903 A-94383.1 A-94384.1 HDV2AD-45027.1 1.001 0.934 A-94385.1 A-94386.1 HDV2 AD-45033.1 0.926 0.939A-94387.1 A-94388.1 HDV2 AD-45038.1 0.838 0.876 A-94389.1 A-94390.1 HDV2AD-45043.1 0.721 0.949 A-94391.1 A-94392.1 HDV2 AD-45048.1 0.873 0.927A-94393.1 A-94394.1 HDV2 AD-45053.1 0.415 0.826 A-94395.1 A-94396.1 HDV2AD-45016.1 0.634 0.917 A-94397.1 A-94398.1 HDV3 AD-45022.1 0.943 0.900A-94399.1 A-94400.1 HDV3 AD-45028.1 0.817 0.914 A-94401.1 A-94402.1 HDV3AD-45034.1 0.601 0.890 A-94403.1 A-94404.1 HDV3 AD-45039.1 0.931 0.888A-94405.1 A-94406.1 HDV3 AD-45044.1 0.912 0.905 A-94407.1 A-94408.1 HDV3AD-45049.1 0.976 0.980 A-94409.1 A-94410.1 HDV3 AD-45054.1 0.929 0.896A-94411.1 A-94412.1 HDV3 AD-45017.1 0.977 0.974 A-94413.1 A-94414.1 HDV3AD-45023.1 0.887 0.878 A-94415.1 A-94416.1 HDV3 AD-45035.1 0.915 0.928A-94419.1 A-94420.1 HDV3 AD-45040.1 1.671 0.934 A-94421.1 A-94422.1 HDV3AD-45050.1 0.878 0.963 A-94425.1 A-94426.1 HDV3 AD-45055.1 0.926 0.873A-94427.1 A-94428.1 HDV3 AD-45018.1 0.912 0.976 A-94429.1 A-94430.1 HDV3AD-45024.1 0.873 0.884 A-94431.1 A-94432.1 HDV3 AD-45030.1 0.909 0.937A-94433.1 A-94434.1 HDV3

Example 5. Synthesis and Screening of Additional siRNA Duplexes Targetedto HBV

Additional iRNA molecules targeting the HBV genome were designed andsynthesized as described above. A detailed list of the additionalunmodified HBV sense and antisense strand sequences is shown in Table 14and a detailed list of the modified HBV sense and antisense strandsequences is shown in Table 15.

TABLE 14 Unmodified Sense and Antisense Strand Sequences of HBV dsRNAsSEQ SEQ Duplex Sense Sequence ID Antisense Antisense Sequence ID IDSense ID (5′ to 3′) NO: ID (5′ to 3′) NO: AD-65381 A-130366.9UCGUGGUGGACUUCUCUCA 1563 A-131904.1 UGAGAGAAGUCCACCACGAUU 1574 AD-66019A-130366.9 UCGUGGUGGACUUCUCUCA 1564 A-131904.1 UGAGAGAAGUCCACCACGAUU1575 AD-65375 A-130366.9 UCGUGGUGGACUUCUCUCA 1565 A-130364.7UGAGAGAAGUCCACCACGAUU 1576 AD-65427 A-130441.7 GUGCACUUCGCUUCACCUCUA1566 A-131905.1 UAGAGGUGAAGCGAAGUGCACUU 1577 AD-66110 A-130441.7GUGCACUUCGCUUCACCUCUA 1567 A-131905.1 UAGAGGUGAAGCGAAGUGCACUU 1578AD-65421 A-130441.7 GUGCACUUCGCUUCACCUCUA 1568 A-130442.6UAGAGGUGAAGCGAAGUGCACUU 1579 AD-65407 A-130371.12 CGUGGUGGACUUCUCUCAAUU1569 A-130372.5 AAUUGAGAGAAGUCCACCAGCAG 1580 AD-65377 A-130384.4CGUGGUGGUCTUCUCUAAAUU 1570 A-130748.3 AAUUGAGAGAAGUCCACCAGCUU 1581AD-65409 A-130388.15 GGUGGACUUCUCUCAAUUUUA 1571 A-131906.1UAAAAUUGAGAGAAGUCCACCAC 1582 AD-66111 A-130388.15 GGUGGACUUCUCUCAAUUUUA1572 A-131906.1 UAAAAUUGAGAGAAGUCCACCAC 1583 AD-65403 A-130388.15GGUGGACUUCUCUCAAUUUUA 1573 A-130389.4 UAAAAUUGAGAGAAGUCCACCAC 1584

TABLE 15 Modified Sense and Antisense Strand Sequences of HBV dsRNAs SEQAnti- SEQ Duplex ID sense ID ID Sense ID Sense Sequence (5′ to 3′) NO:ID Antisense Sequence (5′ to 3′) NO: AD- A- uscsguGfgUfGfGfacuucucucaL961585 A- P usGfsagaGfaAfGfuccaCfcAfcgasusu 1596 65381 130366.9 131904.1AD- A- uscsguGfgUfGfGfacuucucucaL96 1586 A- VPusGfsagaGfaAfGfuccaCfcAfcgasusu 1597 66019 130366.9 131904.1 AD- A-uscsguGfgUfGfGfacuucucucaL96 1587 A- usGfsagaGfaAfGfuccaCfcAfcgasusu1598 65375 130366.9 130364.7 AD- A- gsusgcacUfuCfGfCfuucaccucuaL96 1588A- P usAfsgagGfugaagcgAfaGfugcacsusu 1599 65427 130441.7 131905.1 AD- A-gsusgcacUfuCfGfCfuucaccucuaL96 1589 A- VPusAfsgagGfugaagcgAfaGfugcacsusu 1600 66110 130441.7 131905.1 AD- A-gsusgcacUfuCfGfCfuucaccucuaL96 1590 A- usAfsgagGfugaagcgAfaGfugcacsusu1601 65421 130441.7 130442.6 AD- A- csgsugguGfgAfCfUfucucUfCfaauuL961591 A- asAfsuugAfgAfgAfaguCfcAfccagcsasg 1602 65407 130371.12 130372.5AD- A- csgsuggudGgucdTucucuaaauuL96 1592 A-asdAsuugagagdAagudCcaccagcsusu 1603 65377 130384.4 130748.3 AD- A-gsgsuggaCfuUTCfUfcucaAfUfuuuaL96 1593 A- PusAfsaaaUfuGfAfgagaAfgUfccaccsasc 1604 65409 130388.15 131906.1 AD- A-gsgsuggaCfuUTCfUfcucaAfUfuuuaL96 1594 A- VPusAfsaaaUfuGfAfgagaAfgUfccaccsasc 1605 66111 130388.15 131906.1 AD- A-gsgsuggaCfuUTCfUfcucaAfUfuuuaL96 1595 A-usAfsaaaUfuGfAfgagaAfgUfccaccsasc 1606 65403 130388.15 130389.4

A primary single dose screen of these iRNA duplexes was performed usingthe Dual-Glo® Luciferase assay, as described above. The results of thisscreen in Cos7 cells transfected with the indicated HBV iRNAs are shownin Table 16. Data are expressed as percent of mRNA remaining relative tonegative control at 24 hours.

TABLE 16 HBV single dose primary screen In Cos7 cells using Dual-GloLuciferase ® Assay Dual luciferase primary screen DRC % Messageremaining at 24 hr ED50 Duplex ID at 50 nM STDEV at 1 nM STDEV (nM)AD-65381 9.3 0.24 15.6 0.77 0.019 AD-66019 ND ND ND ND ND AD-65375 24.20.36 71.4 0.69 No ED50 AD-65427 28.8 1.60 41.0 1.73 0.117 AD-66110 ND NDND ND ND AD-65421 47.6 3.49 85.5 4.76 No ED50 AD-65407 14.3 0.52 25.32.11 0.038 AD-65377 21.8 0.31 37.9 1.12 0.130 AD-65409 9.5 0.41 13.20.71 0.013 AD-66111 ND ND ND ND ND AD-65403 12.6 0.50 37.2 2.31 0.069ND—not done

These duplexes were also assayed for dose response for silencing viralRNA using the Dual-Glo® Luciferase assay, as described above. The dosesof the duplexes used for these assays were 50 nM, 8.333333333 nM,1.388888889 nM, 0.231481481 nM, 0.038580247 nM, 0.006430041 nM,0.001071674 nM, 0.000178612 nM, 2.97687×10⁻⁵ nM, 4.96145×10⁻⁶ nM,8.26909×10⁻⁷ nM, and 1.37818E×10⁻⁷ nM, which represent a 1 to 6 dilutionof the duplexes starting at 50 nM over 12 doses. The results of thisscreen in Cos7 cells transfected with the indicated HBV iRNAs are shownin Table 17. Data are expressed as percent of mRNA remaining relative tonegative control at 24 hours.

TABLE 17 Dose response screen In Cos7 cells using Dual-Glo Luciferase ®Assay Dual luciferase HBV reporter cells IC50 (nM) at 24 hr Duplex IDAssay 1 Assay 2 Assay 3 Assay 4 Assay 5 Assay 6 Assay 7 Average¹ StdevAD-65381 0.019 ND ND ND ND ND ND 0.019 AD-66019 ND 0.021 0.021 0.0160.026 0.019 0.031 0.022 0.005 AD-65375 UD 0.215 0.149 0.081 0.246 0.1380.276 0.184 0.074 AD-65407 0.038 0.045 0.051 0.021 0.050 0.056 0.0680.047 0.015 AD-65377 0.130 0.029 0.046 0.087 0.096 0.146 0.090 0.0890.042 AD-65409 0.013 ND ND ND ND ND ND 0.013 AD-66111 ND 0.018 0.0130.012 0.018 0.021 0.033 0.019 0.007 AD-65403 0.069 0.044 0.033 0.0390.042 0.046 0.062 0.048 0.013 AD-65427 0.017 ND ND ND ND ND ND 0.117AD-66110 ND 0.238 0.296 0.145 0.157 0.161 ND 0.199 0.065 AD-65421 UD1.219 1.385 2.254 0.799 2.906 ND 1.713 0.852 ¹Averages from 5-7biological replicates run in triplicate ND—not done

The in vitro efficacy and potency of these duplexes were also assayed.In particular, the dose response of the duplexes for silencing viral RNAin transfected HepG2.2.15 and Hep3B cell lysates and for silencing HBsAgin HepG2.2.15 cell supernatants were determined. Cells were transfectedwith 12 separate doses of the duplexes ranging from 50 nM to 1×10⁻⁷ nMand at seventy-two hours after transfection, the level of viral RNA wasdetermined using primer/probe pairs to detect the P ORF and/or the SORF. The level of HBsAg was determined using an ELISA assay.

The results of the P ORF viral RNA silencing in HepG2.2.15 cells usingthe indicated duplexes are provided in Table 18. The results of the SORF viral RNA silencing in HepG2.2.15 cells using the indicated duplexesare provided in Table 19. The results of HBsAg silencing in HepG2.2.15cells are provided in Table 20.

The results of the P ORF viral RNA silencing in Hep3B cells using theindicated duplexes are provided in Table 21.

TABLE 18 Dose response screen In HepG2.2.15 cells Viral RNA silencing inHepG2.2.15 cells P-ORF primer/probe set IC50 (nM) at 72 hr OptimizedAssay Duplex ID Assay Development Assay 1 Assay 2 Assay 3 AD-65381 0.0790.208 ND ND ND ND ND AD-66019 ND ND 0.265 0.010 0.022 0.032 0.023AD-65375 12.3   UD UD UD 0.172 0.257 0.672 AD-65407 0.247 1.0  0.3650.109 0.069 0.103 0.095 AD-65377 1.3  UD 4.9  UD 0.842 0.838 0.615AD-65409 0.436 1.0  ND ND ND ND ND AD-66111 ND ND 0.456 0.030 50 0.294ND AD-65403 9.2  10.4   3.4  UD 0.114 0.384 1.0  AD-65427 0.007 0.018 NDND ND ND ND AD-66110 ND ND 0.012 0.053 0.016 0.010 0.021 AD-65421 0.0690.091 0.034 0.006 0.002 0.003 0.007 ND—not done

TABLE 19 Dose response screen In HepG2.2.15 cells Viral RNA silencing inHepG2.2.15 cells S-ORF primer/probe set IC50 (nM) at 72 hr OptimizedAssay Duplex ID Assay Development Assay 1 Assay 2 Assay 3 AD-65381 0.2520.215 ND ND ND ND ND AD-66019 ND ND 0.245 0.011 0.009 0.016 0.005AD-65375 45    UD UD UD 0.124 0.048 0.056 AD-65407 0.232 0.645 0.5770.015 0.021 0.023 0.016 AD-65377 1.4  8.6  UD UD 0.575 0.483 0.117AD-65409 0.433 0.242 ND ND ND ND ND AD-66111 ND ND 2.1  0.455 ND 0.416ND AD-65403 0.997 0.670 0.668 UD 0.074 0.270 1.1  AD-65427 0.008 0.018ND ND ND ND ND AD-66110 ND ND 0.022 0.050 0.035 0.038 0.020 AD-654210.083 0.097 0.046 0.003 0.003 0.005 0.001 ND—not done

TABLE 20 Dose response screen In HepG2.2.15 cells HBsAg ELISA IC50 (nM)Duplex ID Assay 1 AD-65381 ND AD-66019 0.105 AD-65375 1.2 AD-65407 0.102AD-65377 2.9 AD-65409 ND AD-66111 0.018 AD-65403 0.064 AD-65427 NDAD-66110 0.002 AD-65421 0.008 ND—not done

TABLE 21 Dose response screen In Hep3B cells Hep3B cells screen DRC ED50P-ORF primer/probe set P-ORF P-ORF Duplex ID run 1 run 2 CombinedAD-65381 0.239 0.110 0.194 AD-66019 ND ND ND AD-65375 ND ND ND AD-654270.023 0.006 0.018 AD-66110 ND ND ND AD-65421 ND ND ND AD-65407 0.1401.383 0.527 AD-65377 No ED50 No ED50 No ED50 AD-65409 1.807 3.436 2.905AD-66111 ND ND ND AD-65403 0.511 18.036 5.013 ND—not done

These duplexes were also assayed for in vitro stability using twoassays, a tritosome stability assay and a cytosol stability assay, asdescribed above. The results of these assays are provided in Table 22.

TABLE 22 Twenty-four hour tritosome and cytosol stability assays. Invitro metabolic stability parent remaining at 24 hr % incubationEndo-lysosome Cytosol Duplex ID % AS % SS % AS % SS AD-65381 88 72 68 22AD-66019 ND ND ND ND AD-65375 ND ND ND ND AD-65407 100 120 64 24AD-65377 99 127 80 67 AD-65409 105 120 86 74 AD-66111 ND ND ND NDAD-65403 ND ND ND ND AD-65427 115 80 89 66 AD-66110 ND ND ND ND AD-65421ND ND ND ND

Dose response screens of various combinations of these duplexes werealso performed in HepG2.215 cells. The doses of the duplexes used forthese assays were 50 nM, 8.333333333 nM, 1.388888889 nM, 0.231481481 nM,0.038580247 nM, 0.006430041 nM, 0.001071674 nM, 0.000178612 nM,2.97687×10⁻⁵ nM, 4.96145×10⁻⁶ nM, 8.26909×10⁻⁷ nM, and 1.37818E×10⁻⁷ nM,which represent a 1 to 6 dilution of the duplexes starting at 50 nM over12 doses. At seventy-two hours after transfection of these duplexes, thelevel of viral RNA (P ORF and S ORF) and the level of secreted HBsAgwere determined, as described above. The results of these assays areprovided in Table 23.

TABLE 23 Seventy-two hour HBV single dose screen In HepG2.2.15 cellsS-ORF2 S-ORF2 S-ORF2 P-ORF1 P-ORF1 P-ORF1 S Ag ELISA IC50_A IC50_BIC50_Combine IC50_A IC50_B IC50_Combine ED50 DuplexID (nM) (nM) (nM)(nM) (nM) (nM) (nM) AD-66019/AD-66110 0.0091 0.0017 0.0038 0.0213 0.0020.0076 0.007482 AD-66019/AD-65421 0.0438 0.2371 0.0131 0.0367 0.01060.0204 0.026398 AD-65375/AD-66110 0.0832 1.0896 0.193 0.0377 0.23480.2022 0.004174 AD-65375/AD-65421 0.084 0.0475 0.0708 0.0566 0.03880.0371 0.030822 AD-65407/AD-66110 0.0387 0.001 0.0083 0.0402 0.00180.0116 0.010172 AD-65407/AD-65421 0.0686 0.0062 0.0225 0.0711 0.01770.0396 0.066556 AD-65377/AD-66110 0.0634 0.8267 0.6269 0.0477 0.0730.0618 0.01435 AD-65377/AD-65421 0.1461 0.0468 0.1372 0.1207 0.00880.0451 0.03419 AD-66111/AD-66110 0.0382 0.0094 0.0161 0.0292 0.00270.0088 0.013155 AD-66111/AD-65421 0.1628 0.0919 0.1579 0.1297 0.03960.0722 0.026889 AD-65403/AD-66110 0.0499 0.0094 0.0444 0.0383 0.01640.0348 0.003783 AD-65403/AD-65421 0.1011 0.0007 0.0208 0.1118 0.00310.0297 0.014569

Example 6. Synthesis and In Vitro Screening of Additional siRNA DuplexesTargeting HBV

Additional iRNA molecules targeting the X ORF of the HBV genome weredesigned and synthesized as described above. A detailed list of theadditional unmodified HBV sense and antisense strand sequences is shownin Table 24. A detailed list of the additional modified HBV sense andantisense strand sequences is shown in Table 25.

TABLE 24 Unmodified Sense and Antisense Strand Sequences of HBV dsRNAsSense SEQ Antisense SEQ Duplex Oligo Sence Sequence ID OligoAntisense Sequence ID ID Name (5′ to 3′) NO: Name (5′ to 3′) NO:AD-65776 A-131859.1 UGUGCACUUCGCUUCACCUCU 1607 A-131860.1AGAGGUGAAGCGAAGUGCACACG 1653 AD-65782 A-131877.1 UGCACUUCGCUUCACCUCUGA1608 A-131878.1 UCAGAGGUGAAGCGAAGUGCACA 1654 AD-65792 A-131865.1GUGUGCACUUCGCUUCACCUA 1609 A-131866.1 UAGGUGAAGCGAAGUGCACACGG 1655AD-65781 A-131861.1 CGUGUGCACUUCGCUUCACCU 1610 A-131862.1AGGUGAAGCGAAGUGCACACGGU 1656 AD-64304 A-128443.6 GUGCACUUCGCUUCACCUCUA1611 A-128444.5 UAGAGGUGAAGCGAAGUGCACAC 1657 AD-65771 A-131857.1CCGUGUGCACUUCGCUUCACA 1612 A-131858.1 UGUGAAGCGAAGUGCACACGGUC 1658AD-65758 A-131867.1 CACUUCGCUUCACCUCUGCAA 1613 A-131868.1UUGCAGAGGUGAAGCGAAGUGCA 1659 AD-65777 A-131875.1 ACUUCGCUUCACCUCUGCACA1614 A-131876.1 UGUGCAGAGGUGAAGCGAAGUGC 1660 AD-61567 A-123525.2GGCUGUAGGCAUAAAUUGGUA 1615 A-123526.2 UACCAAUUUAUGCCUACAGCCUC 1661AD-65772 A-131873.1 UUCGCUUCACCUCUGCACGUA 1616 A-131874.1UACGUGCAGAGGUGAAGCGAAGU 1662 AD-65767 A-131871.1 UCGCUUCACCUCUGCACGUCA1617 A-131872.1 UGACGUGCAGAGGUGAAGCGAAG 1663 AD-65763 A-131869.1CUUCGCUUCACCUCUGCACGU 1618 A-131870.1 ACGUGCAGAGGUGAAGCGAAGUG 1664AD-64281 A-128395.3 CCCCGUCUGUGCCUUCUCAUA 1619 A-128396.2UAUGAGAAGGCACAGACGGGGAG 1665 AD-64311 A-128391.3 CCGUCUGUGCCUUCUCAUCUA1620 A-128392.2 UAGAUGAGAAGGCACAGACGGGG 1666 AD-65790 A-131837.1CCAGCACCAUGCAACUUUUUA 1621 A-131838.1 UAAAAAGUUGCAUGGUGCUGGUG 1667AD-65761 A-131841.1 CACCAGCACCAUGCAACUUUU 1622 A-131842.1AAAAGUUGCAUGGUGCUGGUGCG 1668 AD-65786 A-131849.1 CACCAUGCAACUUUUUCACCU1623 A-131850.1 AGGUGAAAAAGUUGCAUGGUGCU 1669 AD-65785 A-131835.1CAAUGUCAACGACCGACCUUA 1624 A-131836.1 UAAGGUCGGUCGUUGACAUUGCA 1670AD-65787 A-131863.1 CGCUUCACCUCUGCACGUCGA 1625 A-131864.1UCGACGUGCAGAGGUGAAGCGAA 1671 AD-65770 A-131845.1 ACCUUGAGGCAUACUUCAAAG1626 A-131846.1 CUUUGAAGUAUGCCUCAAGGUCG 1672 AD-65766 A-131843.1CCGACCUUGAGGCAUACUUCA 1627 A-131844.1 UGAAGUAUGCCUCAAGGUCGGUC 1673AD-61555 A-123521.2 GACCUUGAGGCAUACUUCAAA 1628 A-123522.2UUUGAAGUAUGCCUCAAGGUCGG 1674 AD-65762 A-131855.1 ACCGACCUUGAGGCAUACUUA1629 A-131856.1 UAAGUAUGCCUCAAGGUCGGUCG 1675 AD-65755 A-131827.1UCGCAUGGAGACCACCGUGAA 1630 A-131828.1 UUCACGGUGGUCUCCAUGCGACG 1676AD-65788 A-131811.1 UUACAUAAGAGGACUCUUGGA 1631 A-131812.1UCCAAGAGUCCUCUUAUGUAAGA 1677 AD-65768 A-131803.1 UCUUACAUAAGAGGACUCUUA1632 A-131804.1 UAAGAGUCCUCUUAUGUAAGACC 1678 AD-61561 A-123523.2ACUUCAAAGACUGUUUGUUUA 1633 A-123524.2 UAAACAAACAGUCUUUGAAGUAU 1679AD-65764 A-131801.1 UACUUCAAAGACUGUUUGUUU 1634 A-131802.1AAACAAACAGUCUUUGAAGUAUG 1680 AD-65753 A-131799.1 AUACUUCAAAGACUGUUUGUU1635 A-131800.1 AACAAACAGUCUUUGAAGUAUGC 1681 AD-65765 A-131817.1UUGUUUAAAGACUGGGAGGAA 1636 A-131818.1 UUCCUCCCAGUCUUUAAACAAAC 1682AD-65769 A-131819.1 GCAUACUUCAAAGACUGUUUA 1637 A-131820.1UAAACAGUCUUUGAAGUAUGCCU 1683 AD-65759 A-131815.1 CAAAGACUGUUUGUUUAAAGA1638 A-131816.1 UCUUUAAACAAACAGUCUUUGAA 1684 AD-65774 A-131831.1AGACUGUUUGUUUAAAGACUA 1639 A-131832.1 UAGUCUUUAAACAAACAGUCUUU 1685AD-65778 A-131807.1 GUUUGUUUAAAGACUGGGAGA 1640 A-131808.1UCUCCCAGUCUUUAAACAAACAG 1686 AD-65773 A-131805.1 GGGGGAGGAGAUUAGAUUAAA1641 A-131806.1 UUUAAUCUAAUCUCCUCCCCCAA 1687 AD-65789 A-131825.1GGGGAGGAGAUUAGAUUAAAG 1642 A-131826.1 CUUUAAUCUAAUCUCCUCCCCCA 1688AD-65783 A-131809.1 GUUGGGGGAGGAGAUUAGAUU 1643 A-131810.1AAUCUAAUCUCCUCCCCCAACUC 1689 AD-65754 A-131813.1 UUGGGGGAGGAGAUUAGAUUA1644 A-131814.1 UAAUCUAAUCUCCUCCCCCAACU 1690 AD-65779 A-131821.1GGGAGGAGAUUAGAUUAAAGA 1645 A-131822.1 UCUUUAAUCUAAUCUCCUCCCCC 1691AD-65791 A-131851.1 UUAGAUUAAAGGUCUUUGUAA 1646 A-131852.1UUACAAAGACCUUUAAUCUAAUC 1692 AD-65760 A-131829.1 UAGAUUAAAGGUCUUUGUACU1647 A-131830.1 AGUACAAAGACCUUUAAUCUAAU 1693 AD-65784 A-131823.1AUUAGAUUAAAGGUCUUUGUA 1648 A-131824.1 UACAAAGACCUUUAAUCUAAUCU 1694AD-65757 A-131853.1 GAGGAGAUUAGAUUAAAGGUA 1649 A-131854.1UACCUUUAAUCUAAUCUCCUCCC 1695 AD-65775 A-131847.1 GGACUCUUGGACUCUCUGCAA1650 A-131848.1 UUGCAGAGAGUCCAAGAGUCCUC 1696 AD-65780 A-131833.1ACUCUUGGACUCUCUGCAAUA 1651 A-131834.1 UAUUGCAGAGAGUCCAAGAGUCC 1697AD-65756 A-131839.1 AGAUUAAAGGUCUUUGUACUA 1652 A-131840.1UAGUACAAAGACCUUUAAUCUAA 1698

TABLE 25 Modified Sense and Antisense Strand Sequences of HBV dsRNAsAnti- Sense SEQ sense SEQ Duplex Oligo ID Oligo ID ID NameSense Sequence (5′ to 3′) NO: Name Antisense Sequence (5′ to 3′) NO: AD-A- UfsgsUfgCfaCfuUfCfGfcUfuC 1699 A- asGfsaGfgUfgAfaGfcgaAfgUfgCfa 174565776 131859.1 faCfcUfcUfL96 131860.1 Cfascsg AD- A-UfsgsCfaCfuUfcGfCfUfuCfaC 1700 A- usCfsaGfaGfgUfgAfagcGfaAfgUfg 174665782 131877.1 fcUfcUfgAfL96 131878.1 Cfascsa AD- A-GfsusGfuGfcAfcUfUfCfgCfuU 1701 A- usAfsgGfuGfaAfgCfgaaGfuGfcAfc 174765792 131865.1 fcAfcCfuAfL96 131866.1 Afcsgsg AD- A-CfsgsUfgUfgCfaCfUfUfcGfcU 1702 A- asGfsgUfgAfaGfcGfaagUfgCfaCfa 174865781 131861.1 fuCfaCfcUfL96 131862.1 Cfgsgsu AD- A-GfsusGfcAfcUfuCfGfCfuUfcA 1703 A- usAfsgAfgGfuGfaAfgcgAfaGfuGfc 174964304 128443.6 fcCfuCfuAfL96 128444.5 Afcsasc AD- A-CfscsGfuGfuGfcAfCfUfuCfgC 1704 A- usGfsuGfaAfgCfgAfaguGfcAfcAfc 175065771 131857.1 fuUfcAfcAfL96 131858.1 Gfgsusc AD- A-CfsasCfuUfcGfcUfUfCfaCfcU 1705 A- usUfsgCfaGfaGfgUfgaaGfcGfaAfg 175165758 131867.1 fcUfgCfaAfL96 131868.1 Ufgscsa AD- A-AfscsUfuCfgCfuUfCfAfcCfuC 1706 A- usGfsuGfcAfgAfgGfugaAfgCfgAfa 175265777 131875.1 fuGfcAfcAfL96 131876.1 Gfusgsc AD- A-GfsgsCfuGfuAfgGfCfAfuAfaA 1707 A- usAfscCfaAfuUfuAfugcCfuAfcAfg 175361567 123525.2 fuUfgGfuAfL96 123526.2 Cfcsusc AD- A-UfsusCfgCfuUfcAfCfCfuCfuG 1708 A- usAfscGfuGfcAfgAfgguGfaAfgCfg 175465772 131873.1 fcAfcGfuAfL96 131874.1 Afasgsu AD- A-UfscsGfcUfuCfaCfCfUfcUfgC 1709 A- usGfsaCfgUfgCfaGfaggUfgAfaGfc 175565767 131871.1 faCfgUfcAfL96 131872.1 Gfasasg AD- A-CfsusUfcGfcUfuCfAfCfcUfcU 1710 A- asCfsgUfgCfaGfaGfgugAfaGfcGfa 175665763 131869.1 fgCfaCfgUfL96 131870.1 Afgsusg AD- A-CfscsCfcGfuCfuGfUfGfcCfuU 1711 A- usAfsuGfaGfaAfgGfcacAfgAfcGfg 175764281 128395.3 fcUfcAfuAfL96 128396.2 Gfgsasg AD- A-CfscsGfuCfuGfuGfCfCfuUfcU 1712 A- usAfsgAfuGfaGfaAfggcAfcAfgAfc 175864311 128391.3 fcAfuCfuAfL96 128392.2 Gfgsgsg AD- A-CfscsAfgCfaCfcAfUfGfcAfaC 1713 A- usAfsaAfaAfgUfuGfcauGfgUfgCfu 175965790 131837.1 fuUfuUfuAfL96 131838.1 Gfgsusg AD- A-CfsasCfcAfgCfaCfCfAfuGfcA 1714 A- asAfsaAfgUfuGfcAfuggUfgCfuGfg 176065761 131841.1 faCfuUfuUfL96 131842.1 Ufgscsg AD- A-CfsasCfcAfuGfcAfAfCfuUfuU 1715 A- asGfsgUfgAfaAfaAfguuGfcAfuGfg 176165786 131849.1 fuCfaCfcUfL96 131850.1 Ufgscsu AD- A-CfsasAfuGfuCfaAfCfGfaCfcG 1716 A- usAfsaGfgUfcGfgUfcguUfgAfcAfu 176265785 131835.1 faCfcUfuAfL96 131836.1 Ufgscsa AD- A-CfsgsCfuUfcAfcCfUfCfuGfcA 1717 A- usCfsgAfcGfuGfcAfgagGfuGfaAfg 176365787 131863.1 fcGfuCfgAfL96 131864.1 Cfgsasa AD- A-AfscsCfuUfgAfgGfCfAfuAfcU 1718 A- csUfsuUfgAfaGfuAfugcCfuCfaAfg 176465770 131845.1 fuCfaAfaGfL96 131846.1 Gfuscsg AD- A-CfscsGfaCfcUfuGfAfGfgCfaU 1719 A- usGfsaAfgUfaUfgCfcucAfaGfgUfc 176565766 131843.1 faCfuUfcAfL96 131844.1 Gfgsusc AD- A-GfsasCfcUfuGfaGfGfCfaUfaC 1720 A- usUfsuGfaAfgUfaUfgccUfcAfaGfg 176661555 123521.2 fuUfcAfaAfL96 123522.2 Ufcsgsg AD- A-AfscsCfgAfcCfuUfGfAfgGfcA 1721 A- usAfsaGfuAfuGfcCfucaAfgGfuCfg 176765762 131855.1 fuAfcUfuAfL96 131856.1 Gfuscsg AD- A-UfscsGfcAfuGfgAfGfAfcCfaC 1722 A- usUfscAfcGfgUfgGfucuCfcAfuGfc 176865755 131827.1 fcGfuGfaAfL96 131828.1 Gfascsg AD- A-UfsusAfcAfuAfaGfAfGfgAfcU 1723 A- usCfscAfaGfaGfuCfcucUfuAfuGfu 176965788 131811.1 fcUfuGfgAfL96 131812.1 Afasgsa AD- A-UfscsUfuAfcAfuAfAfGfaGfgA 1724 A- usAfsaGfaGfuCfcUfcuuAfuGfuAfa 177065768 131803.1 fcUfcUfuAfL96 131804.1 Gfascsc AD- A-AfscsUfuCfaAfaGfAfCfuGfuU 1725 A- usAfsaAfcAfaAfcAfgucUfuUfgAfa 177161561 123523.2 fuGfuUfuAfL96 123524.2 Gfusasu AD- A-UfsasCfuUfcAfaAfGfAfcUfgU 1726 A- asAfsaCfaAfaCfaGfucuUfuGfaAfg 177265764 131801.1 fuUfgUfuUfL96 131802.1 Ufasusg AD- A-AfsusAfcUfuCfaAfAfGfaCfuG 1727 A- asAfscAfaAfcAfgUfcuuUfgAfaGfu 177365753 131799.1 fuUfuGfuUfL96 131800.1 Afusgsc AD- A-UfsusGfuUfuAfaAfGfAfcUfgG 1728 A- usUfscCfuCfcCfaGfucuUfuAfaAfc 177465765 131817.1 fgAfgGfaAfL96 131818.1 Afasasc AD- A-GfscsAfuAfcUfuCfAfAfaGfaC 1729 A- usAfsaAfcAfgUfcUfuugAfaGfuAfu 177565769 131819.1 fuGfuUfuAfL96 131820.1 Gfcscsu AD- A-CfsasAfaGfaCfuGfUfUfuGfuU 1730 A- usCfsuUfuAfaAfcAfaacAfgUfcUfu 177665759 131815.1 fuAfaAfgAfL96 131816.1 Ufgsasa AD- A-AfsgsAfcUfgUfuUfGfUfuUfaA 1731 A- usAfsgUfcUfuUfaAfacaAfaCfaGfu 177765774 131831.1 faGfaCfuAfL96 131832.1 Cfususu AD- A-GfsusUfuGfuUfuAfAfAfgAfcU 1732 A- usCfsuCfcCfaGfuCfuuuAfaAfcAfa 177865778 131807.1 fgGfgAfgAfL96 131808.1 Afcsasg AD- A-GfsgsGfgGfaGfgAfGfAfuUfaG 1733 A- usUfsuAfaUfcUfaAfucuCfcUfcCfc 177965773 131805.1 faUfuAfaAfL96 131806.1 Cfcsasa AD- A-GfsgsGfgAfgGfaGfAfUfuAfgA 1734 A- csUfsuUfaAfuCfuAfaucUfcCfuCfc 178065789 131825.1 fuUfaAfaGfL96 131826.1 Cfcscsa AD- A-GfsusUfgGfgGfgAfGfGfaGfaU 1735 A- asAfsuCfuAfaUfcUfccuCfcCfcCfa 178165783 131809.1 fuAfgAfuUfL96 131810.1 Afcsusc AD- A-UfsusGfgGfgGfaGfGfAfgAfuU 1736 A- usAfsaUfcUfaAfuCfuccUfcCfcCfc 178265754 131813.1 faGfaUfuAfL96 131814.1 Afascsu AD- A-GfsgsGfaGfgAfgAfUfUfaGfaU 1737 A- usCfsuUfuAfaUfcUfaauCfuCfcUfc 178365779 131821.1 fuAfaAfgAfL96 131822.1 Cfcscsc AD- A-UfsusAfgAfuUfaAfAfGfgUfcU 1738 A- usUfsaCfaAfaGfaCfcuuUfaAfuCfu 178465791 131851.1 fuUfgUfaAfL96 131852.1 Afasusc AD- A-UfsasGfaUfuAfaAfGfGfuCfuU 1739 A- asGfsuAfcAfaAfgAfccuUfuAfaUfc 178565760 131829.1 fuGfuAfcUfL96 131830.1 Ufasasu AD- A-AfsusUfaGfaUfuAfAfAfgGfuC 1740 A- usAfscAfaAfgAfcCfuuuAfaUfcUfa 178665784 131823.1 fuUfuGfuAfL96 131824.1 Afuscsu AD- A-GfsasGfgAfgAfuUfAfGfaUfuA 1741 A- usAfscCfuUfuAfaUfcuaAfuCfuCfc 178765757 131853.1 faAfgGfuAfL96 131854.1 Ufcscsc AD- A-GfsgsAfcUfcUfuGfGfAfcUfcU 1742 A- usUfsgCfaGfaGfaGfuccAfaGfaGfu 178865775 131847.1 fcUfgCfaAfL96 131848.1 Cfcsusc AD- A-AfscsUfcUfuGfgAfCfUfcUfcU 1743 A- usAfsuUfgCfaGfaGfaguCfcAfaGfa 178965780 131833.1 fgCfaAfuAfL96 131834.1 Gfuscsc AD- A-AfsgsAfuUfaAfaGfGfUfcUfuU 1744 A- usAfsgUfaCfaAfaGfaccUfuUfaAfu 179065756 131839.1 fgUfaCfuAfL96 131840.1 Cfusasa

A single dose screen of these duplexes was performed in Cos7 cells at 1nm and 50 nm using the Dual-Glo® Luciferase assay described above. Theresults of the assays are provided in Table 26.

TABLE 26 HBV single dose screen using Dual-Glo Luciferase ® Assay DuplexID 50 nM STDEV 1 nM STDEV AD-65776 20.11 4.21 40.79 1.89 AD-65782 26.313.10 61.07 9.16 AD-65792 43.31 5.24 61.09 6.02 AD-65781 25.77 3.66 39.632.87 AD-64304 18.87 1.26 29.72 3.37 AD-65771 17.16 1.78 37.55 2.20AD-65758 31.74 8.26 65.77 11.05 AD-65777 59.76 11.15 77.63 5.14 AD-6156717.69 5.29 26.45 5.66 AD-65772 58.07 9.67 75.66 4.92 AD-65767 29.65 1.6039.64 4.36 AD-65763 25.10 5.77 47.78 9.99 AD-64281 39.07 6.80 51.46 4.19AD-64311 20.51 1.96 37.80 3.53 AD-65790 50.41 7.00 70.30 1.95 AD-6576113.30 4.38 21.14 3.49 AD-65786 12.45 3.51 22.62 0.33 AD-65785 36.87 6.0451.49 4.18 AD-65787 27.97 5.73 48.18 7.65 AD-65770 22.67 5.39 41.48 8.52AD-65766 31.44 3.35 50.25 0.45 AD-61555 18.43 10.83 22.61 0.57 AD-6576218.87 4.86 34.94 4.81 AD-65755 47.03 9.38 83.19 9.68 AD-65788 35.8510.13 58.07 4.78 AD-65768 24.02 2.49 28.55 2.53 AD-61561 8.11 1.29 14.262.27 AD-65764 16.89 3.99 29.10 1.03 AD-65753 19.10 2.87 29.79 5.26AD-65765 55.40 10.72 76.93 8.79 AD-65769 19.24 4.47 23.18 2.54 AD-6575948.86 4.81 87.31 13.75 AD-65774 102.27 12.33 100.79 3.24 AD-65778 64.392.60 80.67 2.59 AD-65773 72.64 7.87 80.80 4.83 AD-65789 73.59 4.35 94.723.32 AD-65783 54.41 7.15 84.46 4.32 AD-65754 62.51 4.12 102.63 21.42AD-65779 47.40 7.51 76.20 2.05 AD-65791 12.09 0.70 19.19 3.46 AD-6576013.50 4.84 25.37 2.09 AD-65784 19.84 1.27 31.04 3.49 AD-65757 22.66 3.9724.50 5.81 AD-65775 47.78 3.30 58.81 3.05 AD-65780 29.10 2.87 42.85 2.73AD-65756 10.49 1.62 19.95 2.58

Based on these assays, RNAi agents targeting five sites in the HBV X ORF(nucleotides 1551, 1577, 1580, 1806, and 1812 of GenBank Accession No.NC_003977.1) were selected for lead optimization and additional agentswere designed and synthesized. These additional agents are evaluated inin vitro assays as described above. A detailed list of the additionalunmodified sense and antisense strand sequences targeting the HBV X ORFis shown in Table 27. A detailed list of the additional modified senseand antisense strand sequences targeting the HBV X ORF is shown in Table28.

These iRNA agents were also assessed for in vivo efficacy using anAAV-HBV mouse model (see, e.g., Yang, et al. (2014) Cell and Mol Immunol11:71). This mouse model exhibits sustained HBV viremia after infectionwith a recombinant adeno-associated virus (AAV) carrying a replicableHBV genome. Liver expression of the HBV gene in these mice mimics HBVinfection in humans and these mice exhibit significant liverinflammation and liver damage, manifested by increased ALT levels,fibrosis and steatosis.

These AAV-HBV mice were subcutaneously administered a single 3 mg/kgdose of AD-66808, AD-66809, AD-66810, AD-66811, AD-66812, AD-66813,AD-66814, AD-66815, AD-66816, and AD-66817 and the level of HBsAg wasdetermined in the serum of the animals pre-dose, and at day 14/15post-dose. The results of these experiments are provided in Table 29 anddemonstrate that serum levels of HBsAg are decrease following a singleadministration of these agents. Table 29 also provides the results of asingle dose screen in Cos7 cells transfected with the indicated HBViRNAs using the Dual-Glo® Luciferase assay, as described above, for thesame RNAi agents. Data are expressed as percent of mRNA remainingrelative to negative control at 24 hours.

TABLE 27 Unmodified HBV X ORF Sense and Antisense Sequences. SEQ SEQDuplex Sense Sequence ID Antisense Sequence ID ID (5′ to 3′) NO:(5′ to 3′) NO: AD-66808 GUCUGUGCCUUCUCAUCUA 1791 UAGAUGAGAAGGCACAGACUU1801 AD-66809 GUCUGUGCCUUCUCAUCUA 1792 UAGAUGAGAAGGCACAGACUU 1802AD-66810 GUGUGCACUUCGCUUCACA 1793 UGUGAAGCGAAGUGCACACUU 1803 AD-66811GUGUGCACUUCGCUUCACA 1794 UGUGAAGCGAAGUGCACACUU 1804 AD-66812UGUGCACUUCGCUUCACCUCU 1795 AGAGGUGAAGCGAAGUGCACAUU 1805 AD-66813UGUGCACUUCGCUUCACCUCU 1796 AGAGGUGAAGCGAAGUGCACAUU 1806 AD-66814CACCAGCACCAUGCAACUUUU 1797 AAAAGUUGCAUGGUGCUGGUGUU 1807 AD-66815CACCAGCACCAUGCAACUUUU 1798 AAAAGUUGCAUGGUGCUGGUGUU 1808 AD-66816CACCAUGCAACUUUUUCACCU 1799 AGGUGAAAAAGUUGCAUGGUGUU 1809 AD-66817CACCAUGCAACUUUUUCACCU 1800 AGGUGAAAAAGUUGCAUGGUGUU 1810

Table 28 Modified HBV X ORF Sense and Antisense Sequences. SEQ SEQDuplex ID ID ID Sense Segue nce (5′ to 3′) NO:Antisense Sequence (5′ to 3′) NO: AD-66808 gsuscuGfuGfCfCfuucucaucuaL961811 usAfsgauGfaGfAfaggcAfcAfgacsusu 1821 AD-66809gsuscuGfuGfCfCfuucucaucuaL96 1812 UfsAfsgauGfaGfAfaggcAfcAfgacsusu 1822AD-66810 gsusguGfcAfCfUfucgcuucacaL96 1813usGfsugaAfgCfGfaaguGfcAfcacsusu 1823 AD-66811gsusguGfcAfCfUfucgcuucacaL96 1814 UfsGfsugaAfgCfGfaaguGfcAfcacsusu 1824AD-66812 usgsugcaCfuUfCfGfcuucaccucuL96 1815asGfsaggUfgAfAfgcgaAfgUfgcacasusu 1825 AD-66813usgsugcaCfuUfCfGfcuucaccucuL96 1816 AfsGfsaggUfgAfAfgcgaAfgUfgcacasusu1826 AD-66814 csasccagCfaCfCfAfugcaacuuuuL96 1817asAfsaagUfuGfCfauggUfgCfuggugsusu 1827 AD-66815csasccagCfaCfCfAfugcaacuuuuL96 1818 AfsAfsaagUfuGfCfauggUfgCfuggugsusu1828 AD-66816 csasccauGfcAfAfCfuuuuucaccuL96 1819asGfsgugAfaAfAfaguuGfcAfuggugsusu 1829 AD-66817csasccauGfcAfAfCfuuuuucaccuL96 1820 AfsGfsgugAfaAfAfaguuGfcAfuggugsusu1830

TABLE 29 Site In vitro IC₅₀ Log₁₀ HBsAg KD (# vRNA¹) Duplex ID Luc HBV(nM) In Vivo @3 mg/kg 1551 AD-66808 0.187 2.4 (4) AD-66809 0.014 1.461577 AD-66810 0.290 1.7 (4) AD-66811 0.029 1.3 1580 AD-66812 0.795 2.19(4) AD-66813 0.074 >>1.14 1806 AD-66814 0.0002 1.5 (4) AD-668150.0001 >>1.56 1812 AD-66816 0.047 1.61 (4) AD-66817 0.0001 1.60 ¹Numberof viral RNAs silenced

Example 6. In Vivo Screening of iRNA Duplexes

A subset of lead iRNA agents was assessed for in vivo efficacy using theAAV-HBV mouse model described above. AAV-HBV mice were administered asingle 3 mg/kg dose of AD-66019, AD-65375, AD-65047, AD-65377, AD-66111,AD-65421, or AD-66110 and the level of HBsAg was determined in the serumof the animals pre-dose, and at days 5 and 10 post-dose. As a control,AAV-HBV mice were administered a 3 mg/kg dose of a dsRNA targetingmouse/rat transtherytin (mrTTR). The results of these experimentsdemonstrate that serum levels of HBsAg are decreased following a singleadministration of these agents.

The percent of pre-dose HBsAg remaining at days 5 and 10 was determinedin these animals following administration of a single 3 mg/kg dose. Asignificant decrease in HBsA at both days 5 and 10 after administrationwere observed in at least AD-65375, AD-65403, AD-65407, AD-65421,AD-66019, and AD-66111.

Based, at least in part, on the results of the in vitro and in vivoassays described above, AD-65403, which silences 3 HBV RNAs, andAD-66810, which silences the X gene, were selected for further analysisfor use in a monotherapy or in a combination therapy of HBV.

In the AAV-HBV mouse model of HBV infection, a single 3 mg/kg dose ofAD-65403 achieved potent and specific knockdown of HBsAg. In particular,a single 3 mg/kg subcutaneous dose of AD-65403 achieved up to a 3.9log₁₀ reduction in HBsAg levels, with a mean HBsAg reduction of 1.8log₁₀ 5-10 days after a single dose.

In the AAV-HBV mouse model of HBV infection, a single 0.3 mg/kg, 1mg/kg, 3 mg/kg, or 9 mg/kg dose of AD-66810 achieved potent and specificknockdown of HBsAg, especially at the higher doses of AD-66810.

In the AAV-HBV mouse model of HBV infection, AD-66810 administered inthree weekly 3 mg/kg doses, achieved potent and specific knockdown ofHBsAg for a period of greater than 4 months.

Example 7. In Vitro Screening of Additional iRNA Duplexes Targeting HDVIn Vitro Screening: Cell Culture and Transfections:

Cos7 cells (ATCC, Manassas, Va.) were grown to near confluence at 37° C.in an atmosphere of 5% CO₂ in DMEM (ATCC) supplemented with 10% FBS,before being released from the plate by trypsinization. Dual-Glo®Luciferase constructs generated in the psiCHECK2 plasmid containing HDVAY633627 derived sequences (Table 30) were transfected intoapproximately 4×10³ cells using Lipofectamine 2000 (Invitrogen, CarlsbadCalif. cat #11668-019). For each well of a 384 well plate, 0.15 μl ofLipofectamine, 4 ng of vector, and an appropriate amount of one of themodified siRNA agents provided in Table 32 were mixed in 15 μl finalvolume of Opti-MEM and allowed to complex at room temperature for 15minutes. Subsequently the mixture was then supplemented with thesuspention of cells (35 ul in complete media). Cells were incubated for48 hours before luciferase was measured.

The unmodified sequences of the agents provided in Table 32 are providedin Table 3, which were synthesized as described above.

Dual-Glo® Luciferase Assay

Firefly (transfection control) and Renilla (fused to HBV targetsequence) luciferases were measured using Dual-Glo® Luciferase Assay(Promega, Madison, Wis., # E2980). First, media was removed from cells.Then Firefly luciferase activity was measured by adding a mixture of 10μl of the complete media and 10 μl of Dual-Glo® Luciferase Reagent perwell. The mixture was incubated at room temperature for 10-15 minutesbefore luminescence (500 nm) was measured on a Spectramax (MolecularDevices) to detect the Firefly luciferase signal. Renilla luciferaseactivity was measured by adding 10l of room temperature Dual-Glo® Stop &Glo® Reagent to each well and the plates were incubated for 10-15minutes before luminescence was again measured to determine the Renillaluciferase signal. The Dual-Glo® Stop & Glo® Reagent quenches thefirefly luciferase signal and sustained luminescence for the Renillaluciferase reaction. siRNA activity was determined by normalizing theRenilla (HDV) signal to the Firefly (control) signal within each well.The magnitude of siRNA activity was then assessed relative to cells thatwere transfected with the same vector but were not treated with siRNA(mock). All transfections were done at n=2 or greater. siRNAconcentration sufficient to inhibit relative Renilla luciferaseexpression by 50% (IC₅₀) was determined by the method described above ata range of concentrations (40 fM-10 nM).

The results of the single dose screen are provided in Table 33 and theresults of the dose response screens are provided in Table 34.

TABLE 30 HDV sequences used in luciferase plasmids Vector NameHDV sequence psiCHECK2-GCGATCGCCTCGAACTTGGGCGGCGAGTCCAGCAGTCTCCTCTTTATCAGAAAAGAGTAAGA HDV-GCACTGAGGACTGCCGCCTCTTGTCGAGATGAGCCGGTCCGAGTTGAAGAAGAAGCGCGATGantigenomeGAAGAGAAGATATTCTCGAGAAGTGGGTGAGTGGAAGAAAGAAAGCGGAGGAACTCGAGAGGGATCTCCGGAAGACAAAGAAGAAGATCAAGAAACTTGAGGTCGAAAATCCCTGGCTGGGAAACATCAAAGGAATTCTCGGAAAGAAGGACAGGGATGGAGAGGGGGCTCCCCCGGCGAAGAGGGCCCGGACGGACCAGATGGAGATAGACTCCGGGCCTAGGAAGAGGCCTCTCAGGGGAGGATTCACCGACAGGGAGAGGCAGGATCACCGACGAAGGAAGGCCCTCGAGAACAAGAAGAAGCAGCTAGCCGCGGGAGGGAAGAGCCTGAGCAAGGAGGAGGAAGAGGAACTCGGAAGGTTGACCCGGGAAGACGAGGAAAGGAAAAGAAGAGTAGCCGGCCCGCGGGTTGGGGGTGTGAACCCCCTCGAAGGCGGATCGAGGGGAGCGCCCGGGGGCGGCTTCGTCCCCAGCATGCAAGGAGTCCCGGAGTCCCCCTTCGCTCGGACGGGGGAGGGGCTGGACATAAGAGGAACCCGGGGATTCCCATAGGATATACTCTTCCCATCCGATCCACCCTTCTCTCCCCAGAGTTGTCGTCCCCAGTGAATAAAGCGGGTTTCCACTCACAGGTTCACCGTCTCGCGTCCTTCTTTCCTCTTCGGGTCGGCATGGCATCTCCACCTCCTCGCGGTCCGACCTGGGCATCCGAAGGAGGACGCACGTCCACTCGGATGGCTAAGGGAGAGCCACTTTTCTCTCGATTCTCTATCGGAATCTAGAGAGATTTGTGGGTCCCATTCGCCATTACCGAGGGGACGGTCCCCTCGGAATGTTGCCCAGCCGGCGCCAGCGAGGAGGCTGGGACCATGCCGGCCATCAGGTAAGAAAGGATGGAACGCGGACCCTGCAGAGTGGGGTCCCGCCATTCCTGGGCGACCCTTGGGGGGGGAGTCGGAATCGAGCATCGGGAAGGGCATCCCATGGCTCCACTGGTCCCCGGTGTTCCCAGCACCCCCTCCGGTCACTTTCGAAGGGGGTCCGGGGTCCCGCTAGATGGGGACGATAAGTCGAGTTCCCCGGGATAAGCCTCACTCGTCCCCTCTCGGGGGGCGGAACACCCACCGGCTAGCCCCGTTGCTTTCTTTGCTTTCCTCCTCGCTTCGGTCTCCCCCTACTCCTAGCATCTCCTCCTATCGCTATGGCCTTACTCCTACCGCTCGAAGCGCCTCTGTTCGCTGAAGGGGTCCTCTGGAGGTGATTTCTCTGCTCATCTCCGAGTGTGTTCCTCCCTCTGGTGTTCTCAACCCTTCGGCCGGAGTGCTCTCCAAACTTGGGCGTCGGGCCTTTCGGATCGGGGGGGCCCCCCCTTCTCTTCCATCTGTCCTCTTTCCCCTTCCGAGATGTCTCCAGCGTTATGGGGAAAGCTTCCGACTCTTGTATTCTCTTTTGGCCTTCTTGGGAGACATCTCCTCGGCGTTCCAATACTCTTTACCACTTTACCCCTCTCGGGCACTGATCCTTCCCCCGCGGACTCTTCGCTCGGAATTGGCCCATGGCGGCCGC (SEQ ID NO: 1835) psiCHECK2-GCGATCGCCATGGGCCAATTCCGAGCGAAGAGTCCGCGGGGGAAGGATCAGTGCCCGAGAGGHDV-genomeGGTAAAGTGGTAAAGAGTATTGGAACGCCGAGGAGATGTCTCCCAAGAAGGCCAAAAGAGAATACAAGAGTCGGAAGCTTTCCCCATAACGCTGGAGACATCTCGGAAGGGGAAAGAGGACAGATGGAAGAGAAGGGGGGGCCCCCCCGATCCGAAAGGCCCGACGCCCAAGTTTGGAGAGCACTCCGGCCGAAGGGTTGAGAACACCAGAGGGAGGAACACACTCGGAGATGAGCAGAGAAATCACCTCCAGAGGACCCCTTCAGCGAACAGAGGCGCTTCGAGCGGTAGGAGTAAGGCCATAGCGATAGGAGGAGATGCTAGGAGTAGGGGGAGACCGAAGCGAGGAGGAAAGCAAAGAAAGCAACGGGGCTAGCCGGTGGGTGTTCCGCCCCCCGAGAGGGGACGAGTGAGGCTTATCCCGGGGAACTCGACTTATCGTCCCCATCTAGCGGGACCCCGGACCCCCTTCGAAAGTGACCGGAGGGGGTGCTGGGAACACCGGGGACCAGTGGAGCCATGGGATGCCCTTCCCGATGCTCGATTCCGACTCCCCCCCCAAGGGTCGCCCAGGAATGGCGGGACCCCACTCTGCAGGGTCCGCGTTCCATCCTTTCTTACCTGATGGCCGGCATGGTCCCAGCCTCCTCGCTGGCGCCGGCTGGGCAACATTCCGAGGGGACCGTCCCCTCGGTAATGGCGAATGGGACCCACAAATCTCTCTAGATTCCGATAGAGAATCGAGAGAAAAGTGGCTCTCCCTTAGCCATCCGAGTGGACGTGCGTCCTCCTTCGGATGCCCAGGTCGGACCGCGAGGAGGTGGAGATGCCATGCCGACCCGAAGAGGAAAGAAGGACGCGAGACGGTGAACCTGTGAGTGGAAACCCGCTTTATTCACTGGGGACGACAACTCTGGGGAGAGAAGGGTGGATCGGATGGGAAGAGTATATCCTATGGGAATCCCCGGGTTCCTCTTATGTCCAGCCCCTCCCCCGTCCGAGCGAAGGGGGACTCCGGGACTCCTTGCATGCTGGGGACGAAGCCGCCCCCGGGCGCTCCCCTCGATCCGCCTTCGAGGGGGTTCACACCCCCAACCCGCGGGCCGGCTACTCTTCTTTTCCTTTCCTCGTCTTCCCGGGTCAACCTTCCGAGTTCCTCTTCCTCCTCCTTGCTCAGGCTCTTCCCTCCCGCGGCTAGCTGCTTCTTCTTGTTCTCGAGGGCCTTCCTTCGTCGGTGATCCTGCCTCTCCCTGTCGGTGAATCCTCCCCTGAGAGGCCTCTTCCTAGGCCCGGAGTCTATCTCCATCTGGTCCGTCCGGGCCCTCTTCGCCGGGGGAGCCCCCTCTCCATCCCTGTCCTTCTTTCCGAGAATTCCTTTGATGTTTCCCAGCCAGGGATTTTCGACCTCAAGTTTCTTGATCTTCTTCTTTGTCTTCCGGAGATCCCTCTCGAGTTCCTCCGCTTTCTTTCTTCCACTCACCCACTTCTCGAGAATATCTTCTCTTCCATCGCGCTTCTTCTTCAACTCGGACCGGCTCATCTCGACAAGAGGCGGCAGTCCTCAGTGCTCTTACTCTTTTCTGATAAAGAGGAGACTGCTGGACTCGCCGCCCAAGTTCGAGGCGGCCGC (SEQ ID NO: 1836) psiCHECK2-GCGATCGCGAAGACAAAGAAGAAGATCAAGAAACTTGAGGTCGAAAATCCCTGGCTGGGAAA HDV-CATCAAAGGAATTCTCGGAAAGAAGGACAGGGATGGCGGCCGC (SEQ ID NO: 1837) antigenomehot spot psiCHECK2-GCGATCGCCATCCCTGTCCTTCTTTCCGAGAATTCCTTTGATGTTTCCCAGCCAGGGATTTTHDV-genome CGACCTCAAGTTTCTTGATCTTCTTCTTTGTCTTCGCGGCCGC (SEQ ID NO: 1838)hot spot

TABLE 31 Unmodified Sense and Antisense Strand Sequences of HDV dsRNAsSense SEQ Antisense SEQ Oligo Senses Sequence ID OligoAntisense Sequence ID Duple Name Name (5′ to 3′) NO: Name (5′ to 3′) NO:AD-67176.1 A-134242.1 UCCCAAGAAGGCCAAAAGAGA 1839 A-134243.1UCUCUUUUGGCCUUCUUGGGAGA 2017 AD-67177.1 A-134244.1 AGUUUCUUGAUCUUCUUCUUU1840 A-134245.1 AAAGAAGAAGAUCAAGAAACUUG 2018 AD-67178.1 A-134246.1UCUUUUCUGAUAAAGAGGAGA 1841 A-134247.1 UCUCCUCUUUAUCAGAAAAGAGU 2019AD-67179.1 A-134248.1 AGUGGUAAAGAGUAUUGGAAA 1842 A-134249.1UUUCCAAUACUCUUUACCACUUU 2020 AD-67180.1 A-134250.1 CUCAAGUUUCUUGAUCUUCUU1843 A-134251.1 AAGAAGAUCAAGAAACUUGAGGU 2021 AD-67181.1 A-134252.1AAGGCCAAAAGAGAAUACAAA 1844 A-134253.1 UUUGUAUUCUCUUUUGGCCUUCU 2022AD-67182.1 A-134254.1 GACCUCAAGUUUCUUGAUCUU 1845 A-134255.1AAGAUCAAGAAACUUGAGGUCGA 2023 AD-67183.1 A-134256.1 AGGCCAAAAGAGAAUACAAGA1846 A-134257.1 UCUUGUAUUCUCUUUUGGCCUUC 2024 AD-67184.1 A-134258.1CGAUAGAGAAUCGAGAGAAAA 1847 A-134259.1 UUUUCUCUCGAUUCUCUAUCGGA 2025AD-67185.1 A-134260.1 GAUAGAGAAUCGAGAGAAAAG 1848 A-134261.1CUUUUCUCUCGAUUCUCUAUCGG 2026 AD-67186.1 A-134262.1 ACCCACAAAUCUCUCUAGAUU1849 A-134263.1 AAUCUAGAGAGAUUUGUGGGUCC 2027 AD-67187.1 A-134264.1GGAUGGGAAGAGUAUAUCCUA 1850 A-134265.1 UAGGAUAUACUCUUCCCAUCCGA 2028AD-67188.1 A-134266.1 CCGAUAGAGAAUCGAGAGAAA 1851 A-134267.1UUUCUCUCGAUUCUCUAUCGGAA 2029 AD-67189.1 A-134268.1 UCCGAUAGAGAAUCGAGAGAA1852 A-134269.1 UUCUCUCGAUUCUCUAUCGGAAU 2030 AD-67190.1 A-134270.1UGAGUGGAAACCCGCUUUAUU 1853 A-134271.1 AAUAAAGCGGGUUUCCACUCACA 2031AD-67191.1 A-134272.1 CCUUCUUUCCGAGAAUUCCUU 1854 A-134273.1AAGGAAUUCUCGGAAAGAAGGAC 2032 AD-67192.1 A-134274.1 CUGUGAGUGGAAACCCGCUUU1855 A-134275.1 AAAGCGGGUUUCCACUCACAGGU 2033 AD-67193.1 A-134276.1GACCCACAAAUCUCUCUAGAU 1856 A-134277.1 AUCUAGAGAGAUUUGUGGGUCCC 2034AD-67194.1 A-134278.1 GCUGGGAAACAUCAAAGGAAU 1857 A-134279.1AUUCCUUUGAUGUUUCCCAGCCA 2035 AD-67195.1 A-134280.1 ACAGUUGGAAGGCUCAAGGAG1858 A-134281.1 UGUCAACCUUCCGAGUUCCUCUU 2036 AD-67196.1 A-134282.1AAGGAGGAGGAACGAGUCCGA 1859 A-134283.1 UUCCUCCUCCUUGCUCAGGCUCU 2037AD-67197.1 A-134284.1 UCUCCUCUUUAUCAGAAAAGA 1860 A-134285.1UCUUUUCUGAUAAAGAGGAGACU 2038 AD-67198.1 A-134286.1 UCCAGCAGUCUCCUCUUUAUA1861 A-134287.1 UAUAAAGAGGAGACUGCUGGACU 2039 AD-67199.1 A-134288.1AAAGAAGAAGAUCAAGAAACU 1862 A-134289.1 AGUUUCUUGAUCUUCUUCUUUGU 2040AD-67200.1 A-134290.1 AAGAAGAAGAUCAAGAAACUU 1863 A-134291.1AAGUUUCUUGAUCUUCUUCUUUG 2041 AD-67201.1 A-134292.1 GAGGUCGAAAAUCCCUGGCUA1864 A-134293.1 UAGCCAGGGAUUUUCGACCUCAA 2042 AD-67202.1 A-134294.1CCCUGGCUGGGAAACAUCAAA 1865 A-134295.1 UUUGAUGUUUCCCAGCCAGGGAU 2043AD-67203.1 A-134296.1 CUGGCUGGGAAACAUCAAAGA 1866 A-134297.1UCUUUGAUGUUUCCCAGCCAGGG 2044 AD-67204.1 A-134298.1 UGGCUGGGAAACAUCAAAGGA1867 A-134299.1 UCCUUUGAUGUUUCCCAGCCAGG 2045 AD-67205.1 A-134300.1GGCUGGGAAACAUCAAAGGAA 1868 A-134301.1 UUCCUUUGAUGUUUCCCAGCCAG 2046AD-67206.1 A-134302.1 UCAAAGGAAUUCUCGGAAAGA 1869 A-134303.1UCUUUCCGAGAAUUCCUUUGAUG 2047 AD-67207.1 A-134304.1 UGGGAAACAUCAAAGGAAUUA1870 A-134305.1 UAAUUCCUUUGAUGUUUCCCAGC 2048 AD-67208.1 A-134306.1GGAAACAUCAAAGGAAUUCUA 1871 A-134307.1 UAGAAUUCCUUUGAUGUUUCCCA 2049AD-67209.1 A-134308.1 GAAACAUCAAAGGAAUUCUCA 1872 A-134309.1UGAGAAUUCCUUUGAUGUUUCCC 2050 AD-67210.1 A-134310.1 AAACAUCAAAGGAAUUCUCGA1873 A-134311.1 UCGAGAAUUCCUUUGAUGUUUCC 2051 AD-67211.1 A-134312.1CAUCAAAGGAAUUCUCGGAAA 1874 A-134313.1 UUUCCGAGAAUUCCUUUGAUGUU 2052AD-67212.1 A-134314.1 AAAGGAAUUCUCGGAAAGAAA 1875 A-134315.1UUUCUUUCCGAGAAUUCCUUUGA 2053 AD-67213.1 A-134316.1 AAGGAAUUCUCGGAAAGAAGA1876 A-134317.1 UCUUCUUUCCGAGAAUUCCUUUG 2054 AD-67214.1 A-134320.1GGCCCUCGAGAACAAGAAGAA 1877 A-134321.1 UUCUUCUUGUUCUCGAGGGCCUU 2055AD-67215.1 A-134322.1 GCCCUCGAGAACAAGAAGAAA 1878 A-134323.1UUUCUUCUUGUUCUCGAGGGCCU 2056 AD-67216.1 A-134324.1 CCCUCGAGAACAAGAAGAAGA1879 A-134325.1 UCUUCUUCUUGUUCUCGAGGGCC 2057 AD-67217.1 A-134326.1GGAAAGGAAAAGAAGAGUAGA 1880 A-134327.1 UCUACUCUUCUUUUCCUUUCCUC 2058AD-67218.1 A-134328.1 AAGGAAAAGAAGAGUAGCCGA 1881 A-134329.1UCGGCUACUCUUCUUUUCCUUUC 2059 AD-67219.1 A-134330.1 GGGGGUGUGAACCCCCUCGAA1882 A-134331.1 UUCGAGGGGGUUCACACCCCCAA 2060 AD-67220.1 A-134332.1GGGUGUGAACCCCCUCGAAGA 1883 A-134333.1 UCUUCGAGGGGGUUCACACCCCC 2061AD-67250.1 A-134346.1 AGUUUCUUGAUCUUCUUCUUU 1884 A-134347.1AAAGAAGAAGAUCAAGAAACUUG 2062 AD-67251.1 A-134348.1 UCUUUUCUGAUAAAGAGGAGA1885 A-134349.1 UCUCCUCUUUAUCAGAAAAGAGU 2063 AD-67252.1 A-134350.1AGUGGUAAAGAGUAUUGGAAA 1886 A-134351.1 UUUCCAAUACUCUUUACCACUUU 2064AD-67253.1 A-134352.1 CUCAAGUUUCUUGAUCUUCUU 1887 A-134353.1AAGAAGAUCAAGAAACUUGAGGU 2065 AD-67254.1 A-134354.1 AAGGCCAAAAGAGAAUACAAA1888 A-134355.1 UUUGUAUUCUCUUUUGGCCUUCU 2066 AD-67255.1 A-134358.1AGGCCAAAAGAGAAUACAAGA 1889 A-134359.1 UCUUGUAUUCUCUUUUGGCCUUC 2067AD-67256.1 A-134360.1 CGAUAGAGAAUCGAGAGAAAA 1890 A-134361.1UUUUCUCUCGAUUCUCUAUCGGA 2068 AD-67257.1 A-134362.1 GAUAGAGAAUCGAGAGAAAAG1891 A-134363.1 CUUUUCUCUCGAUUCUCUAUCGG 2069 AD-67258.1 A-134364.1ACCCACAAAUCUCUCUAGAUU 1892 A-134365.1 AAUCUAGAGAGAUUUGUGGGUCC 2070AD-67259.1 A-134366.1 GGAUGGGAAGAGUAUAUCCUA 1893 A-134367.1UAGGAUAUACUCUUCCCAUCCGA 2071 AD-67260.1 A-134368.1 CCGAUAGAGAAUCGAGAGAAA1894 A-134369.1 UUUCUCUCGAUUCUCUAUCGGAA 2072 AD-67261.1 A-134370.1UCCGAUAGAGAAUCGAGAGAA 1895 A-134371.1 UUCUCUCGAUUCUCUAUCGGAAU 2073AD-67262.1 A-134372.1 UGAGUGGAAACCCGCUUUAUU 1896 A-134373.1AAUAAAGCGGGUUUCCACUCACA 2074 AD-67263.1 A-134374.1 CCUUCUUUCCGAGAAUUCCUU1897 A-134375.1 AAGGAAUUCUCGGAAAGAAGGAC 2075 AD-67264.1 A-134376.1CUGUGAGUGGAAACCCGCUUU 1898 A-134377.1 AAAGCGGGUUUCCACUCACAGGU 2076AD-67265.1 A-134378.1 GACCCACAAAUCUCUCUAGAU 1899 A-134379.1AUCUAGAGAGAUUUGUGGGUCCC 2077 AD-67266.1 A-134380.1 GCUGGGAAACAUCAAAGGAAU1900 A-134381.1 AUUCCUUUGAUGUUUCCCAGCCA 2078 AD-67267.1 A-134382.1ACAGUUGGAAGGCUCAAGGAG 1901 A-134383.1 UGUCAACCUUCCGAGUUCCUCUU 2079AD-67268.1 A-134384.1 AAGGAGGAGGAACGAGUCCGA 1902 A-134385.1UUCCUCCUCCUUGCUCAGGCUCU 2080 AD-67269.1 A-134386.1 UCUCCUCUUUAUCAGAAAAGA1903 A-134387.1 UCUUUUCUGAUAAAGAGGAGACU 2081 AD-67270.1 A-134388.1UCCAGCAGUCUCCUCUUUAUA 1904 A-134389.1 UAUAAAGAGGAGACUGCUGGACU 2082AD-67271.1 A-134390.1 AAAGAAGAAGAUCAAGAAACU 1905 A-134391.1AGUUUCUUGAUCUUCUUCUUUGU 2083 AD-67272.1 A-134392.1 AAGAAGAAGAUCAAGAAACUU1906 A-134393.1 AAGUUUCUUGAUCUUCUUCUUUG 2084 AD-67273.1 A-134394.1GAGGUCGAAAAUCCCUGGCUA 1907 A-134395.1 UAGCCAGGGAUUUUCGACCUCAA 2085AD-67274.1 A-134396.1 CCCUGGCUGGGAAACAUCAAA 1908 A-134397.1UUUGAUGUUUCCCAGCCAGGGAU 2086 AD-67275.1 A-134398.1 CUGGCUGGGAAACAUCAAAGA1909 A-134399.1 UCUUUGAUGUUUCCCAGCCAGGG 2087 AD-67276.1 A-134400.1UGGCUGGGAAACAUCAAAGGA 1910 A-134401.1 UCCUUUGAUGUUUCCCAGCCAGG 2088AD-67277.1 A-134402.1 GGCUGGGAAACAUCAAAGGAA 1911 A-134403.1UUCCUUUGAUGUUUCCCAGCCAG 2089 AD-67278.1 A-134404.1 UCAAAGGAAUUCUCGGAAAGA1912 A-134405.1 UCUUUCCGAGAAUUCCUUUGAUG 2090 AD-67279.1 A-134406.1UGGGAAACAUCAAAGGAAUUA 1913 A-134407.1 UAAUUCCUUUGAUGUUUCCCAGC 2091AD-67280.1 A-134408.1 GGAAACAUCAAAGGAAUUCUA 1914 A-134409.1UAGAAUUCCUUUGAUGUUUCCCA 2092 AD-67281.1 A-134410.1 GAAACAUCAAAGGAAUUCUCA1915 A-134411.1 UGAGAAUUCCUUUGAUGUUUCCC 2093 AD-67282.1 A-134414.1CAUCAAAGGAAUUCUCGGAAA 1916 A-134415.1 UUUCCGAGAAUUCCUUUGAUGUU 2094AD-67283.1 A-134416.1 AAAGGAAUUCUCGGAAAGAAA 1917 A-134417.1UUUCUUUCCGAGAAUUCCUUUGA 2095 AD-67284.1 A-134418.1 AAGGAAUUCUCGGAAAGAAGA1918 A-134419.1 UCUUCUUUCCGAGAAUUCCUUUG 2096 AD-67285.1 A-134420.1ACGAAGGAAGGCCCUCGAGAA 1919 A-134421.1 UUCUCGAGGGCCUUCCUUCGUCG 2097AD-67286.1 A-134422.1 GGCCCUCGAGAACAAGAAGAA 1920 A-134423.1UUCUUCUUGUUCUCGAGGGCCUU 2098 AD-67287.1 A-134424.1 GCCCUCGAGAACAAGAAGAAA1921 A-134425.1 UUUCUUCUUGUUCUCGAGGGCCU 2099 AD-67288.1 A-134426.1CCCUCGAGAACAAGAAGAAGA 1922 A-134427.1 UCUUCUUCUUGUUCUCGAGGGCC 2100AD-67289.1 A-134428.1 GGAAAGGAAAAGAAGAGUAGA 1923 A-134429.1UCUACUCUUCUUUUCCUUUCCUC 2101 AD-67290.1 A-134430.1 AAGGAAAAGAAGAGUAGCCGA1924 A-134431.1 UCGGCUACUCUUCUUUUCCUUUC 2102 AD-67291.1 A-134432.1GGGGGUGUGAACCCCCUCGAA 1925 A-134433.1 UUCGAGGGGGUUCACACCCCCAA 2103AD-67292.1 A-134434.1 GGGUGUGAACCCCCUCGAAGA 1926 A-134435.1UCUUCGAGGGGGUUCACACCCCC 2104 AD-70224.1 A-141079.1 AAACUUGAGGUCGAAAAUA1927 A-141080.1 UAUUUUCGACCUCAAGUUUUU 2105 AD-70225.1 A-141081.1AAACUUGAGGUCGAAAAUA 1928 A-141082.1 UAUUUUCGACCUCAAGUUUUU 2106AD-70226.1 A-141083.1 AAACUUGAGGUCGAAAAUA 1929 A-141084.1UAUUUUCGACCTCAAGUUUUU 2107 AD-70227.1 A-141085.1 AAACUUGAGGUCGAAAAUA1930 A-141084.1 UAUUUUCGACCTCAAGUUUUU 2108 AD-70228.1 A-141086.1AGAAACUUGAGGUCGAAAAUA 1931 A-141087.1 UAUUUUCGACCUCAAGUUUCU 2109AD-70229.1 A-141090.1 GAUCAAGAAACUUGAGGUA 1932 A-141091.1UACCUCAAGUUUCUUGAUCUU 2110 AD-70230.1 A-141092.1 GAUCAAGAAACUUGAGGUA1933 A-141093.1 UACCUCAAGUUTCUUGAUCUU 2111 AD-70231.1 A-141094.1GAUCAAGAAACUUGAGGUA 1934 A-141093.1 UACCUCAAGUUTCUUGAUCUU 2112AD-70232.1 A-141095.1 AAGAUCAAGAAACUUGAGGUA 1935 A-141096.1UACCUCAAGUUUCUUGAUCUU 2113 AD-70233.1 A-141097.1 UCAAGAAACUUGAGGUCGA1936 A-141098.1 UCGACCUCAAGUUUCUUGAUU 2114 AD-70234.1 A-141097.1UCAAGAAACUUGAGGUCGA 1937 A-141099.1 UCGACCUCAAGUUUCUUGAUU 2115AD-70235.1 A-141100.1 UCAAGAAACUUGAGGUCGA 1938 A-141101.1UCGACCUCAAGUUUCUUGAUU 2116 AD-70236.1 A-141102.1 UCAAGAAACUUGAGGUCGA1939 A-141103.1 UCGACCUCAAGTUUCUUGAUU 2117 AD-70237.1 A-141104.1UCAAGAAACUUGAGCUCGA 1940 A-141103.1 UCGACCUCAAGTUUCUUGAUU 2118AD-70238.1 A-141105.1 UCAAGAAACUUGAGGUCGA 1941 A-141106.1UCGACCUCAAGUUUCUUGA 2119 AD-70239.1 A-141107.1 AAGAUCAAGAAACUUGAGA 1942A-141108.1 UCUCAAGUUUCUUGAUCUUUU 2120 AD-70240.1 A-141107.1AAGAUCAAGAAACUUGAGA 1943 A-141109.1 UCUCAAGUUUCUUGAUCUUUU 2121AD-70241.1 A-141110.1 AAGAUCAAGAAACUUGAGA 1944 A-141111.1UCUCAAGUUUCUUGAUCUUUU 2122 AD-70242.1 A-141112.1 AAGAUCAAGAAACUUGAGA1945 A-141113.1 UCUCAAGUUUCTUGAUCUUUU 2123 AD-70243.1 A-141114.1AAGAUCAAGAAACUAGAGA 1946 A-141113.1 UCUCAAGUUUCTUGAUCUUUU 2124AD-70244.1 A-141115.1 AGAAGAUCAAGAAACUUGAGA 1947 A-141116.1UCUCAAGUUUCUUGAUCUUCU 2125 AD-70245.1 A-141117.1 CAUCAAAGGAAUUCUCGGA1948 A-141118.1 UCCGAGAAUUCCUUUGAUGUU 2126 AD-70246.1 A-141119.1CAUCAAAGGAAUUCUCGGA 1949 A-141120.1 UCCGAGAAUUCCUUUGAUGUU 2127AD-70247.1 A-141121.1 CAUCAAAGGAAUUCUCGGA 1950 A-141122.1UCCGAGAAUUCCUUUGAUGUU 2128 AD-70248.1 A-141123.1 CAUCAAAGGAAUUCTCGGA1951 A-141122.1 UCCGAGAAUUCCUUUGAUGUU 2129 AD-70249.1 A-141124.1AACAUCAAAGGAAUUCUCGGA 1952 A-141125.1 UCCGAGAAUUCCUUUGAUGUU 2130AD-70250.1 A-141126.1 AUCAAGAAACUUGAGGUCA 1953 A-141127.1UGACCUCAAGUUUCUUGAUUU 2131 AD-70251.1 A-141126.1 AUCAAGAAACUUGAGGUCA1954 A-141128.1 UGACCUCAAGUUUCUUGAUUU 2132 AD-70252.1 A-141129.1AUCAAGAAACUUGAGGUCA 1955 A-141130.1 UGACCUCAAGUUUCUUGAUUU 2133AD-70253.1 A-141131.1 AUCAAGAAACUUGAGGUCA 1956 A-141132.1UGACCUCAAGUTUCUUGAUUU 2134 AD-70254.1 A-141133.1 AUCAAGAAACUUGACGUCA1957 A-141132.1 UGACCUCAAGUTUCUUGAUUU 2135 AD-70255.1 A-141134.1AGAUCAAGAAACUUGAGGUCA 1958 A-141135.1 UGACCUCAAGUUUCUUGAUCU 2136AD-70256.1 A-141136.1 GAAGAUCAAGAAACUUGAA 1959 A-141137.1UUCAAGUUUCUUGAUCUUCUU 2137 AD-70257.1 A-141138.1 GAAGAUCAAGAAACUUGAA1960 A-141139.1 UUCAAGUUUCUUGAUCUUCUU 2138 AD-70258.1 A-141140.1GAAGAUCAAGAAACUUGAA 1961 A-141141.1 UUCAAGUUUCUTGAUCUUCUU 2139AD-70259.1 A-141142.1 GAAGAUCAAGAAACTUGAA 1962 A-141141.1UUCAAGUUUCUTGAUCUUCUU 2140 AD-70260.1 A-141143.1 AAGAAGAUCAAGAAACUUGAA1963 A-141144.1 UUCAAGUUUCUUGAUCUUCUU 2141 AD-70261.1 A-141145.1CAAAGGAAUUCUCGGAAAG 1964 A-141146.1 CUUUCCGAGAAUUCCUUUGAU 2142AD-70262.1 A-141145.1 CAAAGGAAUUCUCGGAAAG 1965 A-141147.1CUUUCCGAGAAUUCCUUUGAU 2143 AD-70263.1 A-141148.1 CAAAGGAAUUCUCGGAAAG1966 A-141149.1 CUUUCCGAGAAUUCCUUUGAU 2144 AD-70264.1 A-141150.1CAAAGGAAUUCUCGGAAAG 1967 A-141151.1 CUUUCCGAGAATUCCUUUGAU 2145AD-70265.1 A-141152.1 CAAAGGAAUUCUCGCAAAG 1968 A-141151.1CUUUCCGAGAATUCCUUUGAU 2146 AD-70266.1 A-141153.1 AUCAAAGGAAUUCUCGGAAAG1969 A-141154.1 CUUUCCGAGAAUUCCUUUGAU 2147 AD-70267.1 A-141155.1CAAGAAACUUGAGGUCGAA 1970 A-141156.1 UUCGACCUCAAGUUUCUUGAU 2148AD-70268.1 A-141155.1 CAAGAAACUUGAGGUCGAA 1971 A-141157.1UUCGACCUCAAGUUUCUUGAU 2149 AD-70269.1 A-141158.1 CAAGAAACUUGAGGUCGAA1972 A-141159.1 UUCGACCUCAAGUUUCUUGAU 2150 AD-70270.1 A-141160.1CAAGAAACUUGAGGUCGAA 1973 A-141161.1 UUCGACCUCAAGUUUCUUGAU 2151AD-70271.1 A-141162.1 CAAGAAACUUGAGGTCGAA 1974 A-141161.1UUCGACCUCAAGUUUCUUGAU 2152 AD-70272.1 A-141163.1 AUCAAGAAACUUGAGGUCGAA1975 A-141164.1 UUCGACCUCAAGUUUCUUGAU 2153 AD-70273.1 A-141165.1GAAGAAGAUCAAGAAACUU 1976 A-141166.1 AAGUUUCUUGAUCUUCUUCUU 2154AD-70274.1 A-141165.1 GAAGAAGAUCAAGAAACUU 1977 A-141167.1AAGUUUCUUGAUCUUCUUCUU 2155 AD-70275.1 A-141168.1 GAAGAAGAUCAAGAAACUU1978 A-141169.1 AAGUUUCUUGAUCUUCUUCUU 2156 AD-70276.1 A-141170.1GAAGAAGAUCAAGAAACUU 1979 A-141171.1 AAGUUUCUUGATCUUCUUCUU 2157AD-70277.1 A-141172.1 GAAGAAGAUCAAGAAACUU 1980 A-141171.1AAGUUUCUUGATCUUCUUCUU 2158 AD-70278.1 A-141173.1 AAGAAGAAGAUCAAGAAACUU1981 A-141174.1 AAGUUUCUUGAUCUUCUUCUU 2159 AD-70279.1 A-141175.1GGAAACAUCAAAGGAAUUA 1982 A-141176.1 UAAUUCCUUUGAUGUUUCCUU 2160AD-70280.1 A-141175.1 GGAAACAUCAAAGGAAUUA 1983 A-141177.1UAAUUCCUUUGAUGUUUCCUU 2161 AD-70281.1 A-141178.1 GGAAACAUCAAAGGAAUUA1984 A-141179.1 UAAUUCCUUUGAUGUUUCCUU 2162 AD-70282.1 A-141180.1GGAAACAUCAAAGGAAUUA 1985 A-141181.1 UAAUUCCUUUGAUGUUUCUU 2163 AD-70283.1A-141182.1 GGAAACAUCAAAGGAAUUA 1986 A-141183.1 UAAUUCCUUUGAUGUUUCCUU2164 AD-70284.1 A-141184.1 UGGGAAACAUCAAAGGAAUUA 1987 A-141185.1UAAUUCCUUUGAUGUUUCCCA 2165 AD-70285.1 A-134288.1 AAAGAAGAAGAUCAAGAAACU1988 A-141186.1 AGUUUCUUGAUCUUCUUCUUUGU 2166 AD-70286.1 A-141187.1AAAGAAGAAGAUCAAGAAACU 1989 A-141188.1 AGUUUCUUGAUCUUCUUCUUUGU 2167AD-70287.1 A-141189.1 AAAGAAGAAGAUCAAGAAACU 1990 A-141190.1AGUUUCUUGAUCUUCUUCUUUGU 2168 AD-70288.1 A-141191.1 AAAGAAGAAGAUCAAGAAACU1991 A-141190.1 AGUUUCUUGAUCUUCUUCUUUGU 2169 AD-70289.1 A-141192.1AAAGAAGAAGAUCAAGAAACU 1992 A-141193.1 AGUUUCUUGAUCUUCUUCUUU 2170AD-70290.1 A-141194.1 AGAAGAAGAUCAAGAAACU 1993 A-141195.1AGUUUCUUGAUCUUCUUCUUU 2171 AD-70291.1 A-141194.1 AGAAGAAGAUCAAGAAACU1994 A-141196.1 AGUUUCUUGAUCUUCUUCUUU 2172 AD-70292.1 A-141197.1AGAAGAAGAUCAAGAAACU 1995 A-141198.1 AGUUUCUUGAUCUUCUUCUUU 2173AD-70293.1 A-141199.1 AGAAGAAGAUCAAGAAACU 1996 A-141200.1AGUUUCUUGAUCUUCUUCUUU 2174 AD-70294.1 A-141201.1 AGAAGAAGAUCAAGAAACU1997 A-141200.1 AGUUUCUUGAUCUUCUUCUUU 2175 AD-70295.1 A-141202.1AGAAGAAGAUCAAGAAACU 1998 A-141203.1 AGUUUCUUGAUCUUCUUCU 2176 AD-70296.1A-141204.1 AACAUCAAAGGAAUUCUCA 1999 A-141205.1 UGAGAAUUCCUUUGAUGUUUU2177 AD-70297.1 A-141204.1 AACAUCAAAGGAAUUCUCA 2000 A-141206.1UGAGAAUUCCUUUGAUGUUUU 2178 AD-70298.1 A-141207.1 AACAUCAAAGGAAUUCUCA2001 A-141208.1 UGAGAAUUCCUUUGAUGUUUU 2179 AD-70299.1 A-141209.1AACAUCAAAGGAAUUCUCA 2002 A-141210.1 UGAGAAUUCCUTUGAUGUUUU 2180AD-70300.1 A-141211.1 AACAUCAAAGGAAUTCUCA 2003 A-141210.1UGAGAAUUCCUTUGAUGUUUU 2181 AD-70301.1 A-141212.1 AACAUCAAAGGAAUUCUCA2004 A-141213.1 UGAGAAUUCCUUUGAUGUU 2182 AD-70302.1 A-141214.1AGGAAUUCUCGGAAAGAAA 2005 A-141215.1 UUUCUUUCCGAGAAUUCCUUU 2183AD-70303.1 A-141214.1 AGGAAUUCUCGGAAAGAAA 2006 A-141216.1UUUCUUUCCGAGAAUUCCUUU 2184 AD-70304.1 A-141217.1 AGGAAUUCUCGGAAAGAAA2007 A-141218.1 UUUCUUUCCGAGAAUUCCUUU 2185 AD-70305.1 A-141219.1AGGAAUUCUCGGAAAGAAA 2008 A-141220.1 UUUCUUUCCGAGAAUUCCUUU 2186AD-70306.1 A-141221.1 AGGAAUUCUCGGAAAGAAA 2009 A-141220.1UUUCUUUCCGAGAAUUCCUUU 2187 AD-70307.1 A-141222.1 AAAGGAAUUCUCGGAAAGAAA2010 A-141223.1 UUUCUUUCCGAGAAUUCCUUU 2188 AD-70308.1 A-141224.1AAAGGAAUUCUCGGAAAGA 2011 A-141225.1 UCUUUCCGAGAAUUCCUUUUU 2189AD-70309.1 A-141224.1 AAAGGAAUUCUCGGAAAGA 2012 A-141226.1UCUUUCCGAGAAUUCCUUUUU 2190 AD-70310.1 A-141227.1 AAAGGAAUUCUCGGAAAGA2013 A-141228.1 UCUUUCCGAGAAUUCCUUUUU 2191 AD-70311.1 A-141229.1AAAGGAAUUCUCGGAAAGA 2014 A-141230.1 UCUUUCCGAGAAUUCCUUUUU 2192AD-70312.1 A-141231.1 AAAGGAAUUCUCGGAAAGA 2015 A-141230.1UCUUUCCGAGAAUUCCUUUUU 2193 AD-70313.1 A-141232.1 UCAAAGGAAUUCUCGGAAAGA2016 A-141233.1 UCUUUCCGAGAAUUCCUUUGA 2194

TABLE 32 Modified Sense and Antisense Strand Sequences of HDV dsRNAsAnti- Sense SEQ sense SEQ Duplex Oligo ID Oligo ID Name NameSense Sequence (5′3′ NO: Name Antisense Sequence (5′3′ NO: AD- A-uscsccaaGfaAfGfGfccaaaagagaL96 2195 A- usCfsucuUfuuggccuUfcUfugggasgsa2373 67176.1 134242.1 134243.1 AD- A- asgsuuucUfuGfAfUfcuucuucuuuL962196 A- asAfsagaAfgaagaucAfaGfaaacususg 2374 67177.1 134244.1 134245.1AD- A- uscsuuuuCfuGfAfUfaaagaggagaL96 2197 A-usCfsuccUfcuuuaucAfgAfaaagasgsu 2375 67178.1 134246.1 134247.1 AD- A-asgsugguAfaAfGfAfguauuggaaaL96 2198 A- usUfsuccAfauacucuUfuAfccacususu2376 67179.1 134248.1 134249.1 AD- A- csuscaagUfuUfCfUfugaucuucuuL962199 A- asAfsgaaGfaucaagaAfaCfuugagsgsu 2377 67180.1 134250.1 134251.1AD- A- asasggccAfaAfAfGfagaauacaaaL96 2200 A-usUfsuguAfuucucuuUfuGfgccuuscsu 2378 67181.1 134252.1 134253.1 AD- A-gsasccucAfaGfUfUfucuugaucuuL96 2201 A- asAfsgauCfaagaaacUfuGfaggucsgsa2379 67182.1 134254.1 134255.1 AD- A- asgsgccaAfaAfGfAfgaauacaagaL962202 A- usCfsuugUfauucucuUfuUfggccususc 2380 67183.1 134256.1 134257.1AD- A- csgsauagAfgAfAfUfcgagagaaaaL96 2203 A-usUfsuucUfcucgauuCfuCfuaucgsgsa 2381 67184.1 134258.1 134259.1 AD- A-gsasuagaGfaAfUfCfgagagaaaagL96 2204 A- csUfsuuuCfucucgauUfcUfcuaucsgsg2382 67185.1 134260.1 134261.1 AD- A- ascsccacAfaAfUfCfucucuagauuL962205 A- asAfsucuAfgagagauUfuGfuggguscsc 2383 67186.1 134262.1 134263.1AD- A- gsgsauggGfaAfGfAfguauauccuaL96 2206 A-usAfsggaUfauacucuUfcCfcauccsgsa 2384 67187.1 134264.1 134265.1 AD- A-cscsgauaGfaGfAfAfucgagagaaaL96 2207 A- usUfsucuCfucgauucUfcUfaucggsasa2385 67188.1 134266.1 134267.1 AD- A- uscscgauAfgAfGfAfaucgagagaaL962208 A- usUfscucUfcgauucuCfuAfucggasasu 2386 67189.1 134268.1 134269.1AD- A- usgsagugGfaAfAfCfccgcuuuauuL96 2209 A-asAfsuaaAfgcggguuUfcCfacucascsa 2387 67190.1 134270.1 134271.1 AD- A-cscsuucuUfuCfCfGfagaauuccuuL96 2210 A- asAfsggaAfuucucggAfaAfgaaggsasc2388 67191.1 134272.1 134273.1 AD- A- csusgugaGfuGfGfAfaacccgcuuuL962211 A- asAfsagcGfgguuuccAfcUfcacagsgsu 2389 67192.1 134274.1 134275.1AD- A- gsascccaCfaAfAfUfcucucuagauL96 2212 A-asUfscuaGfagagauuUfgUfgggucscsc 2390 67193.1 134276.1 134277.1 AD- A-gscsugggAfaAfCfAfucaaaggaauL96 2213 A- asUfsuccUfuugauguUfuCfccagcscsa2391 67194.1 134278.1 134279.1 AD- A- ascsaguuGfgAfAfGfgcucaaggagL962214 A- usGfsucaAfccuuccgAfgUfuccucsusu 2392 67195.1 134280.1 134281.1AD- A- asasggagGfaGfGfAfacgaguccgaL96 2215 A-usUfsccuCfcuccuugCfuCfaggcuscsu 2393 67196.1 134282.1 134283.1 AD- A-uscsuccuCfuUfUfAfucagaaaagaL96 2216 A- usCfsuuuUfcugauaaAfgAfggagascsu2394 67197.1 134284.1 134285.1 AD- A- uscscagcAfgUfCfUfccucuuuauaL962217 A- usAfsuaaAfgaggagaCfuGfcuggascsu 2395 67198.1 134286.1 134287.1AD- A- asasagaaGfaAfGfAfucaagaaacuL96 2218 A-asGfsuuuCfuugaucuUfcUfucuuusgsu 2396 67199.1 134288.1 134289.1 AD- A-asasgaagAfaGfAfUfcaagaaacuuL96 2219 A- asAfsguuUfcuugaucUfuCfuucuususg2397 67200.1 134290.1 134291.1 AD- A- gsasggucGfaAfAfAfucccuggcuaL962220 A- usAfsgccAfgggauuuUfcGfaccucsasa 2398 67201.1 134292.1 134293.1AD- A- cscscuggCfuGfGfGfaaacaucaaaL96 2221 A-usUfsugaUfguuucccAfgCfcagggsasu 2399 67202.1 134294.1 134295.1 AD- A-csusggcuGfgGfAfAfacaucaaagaL96 2222 A- usCfsuuuGfauguuucCfcAfgccagsgsg2400 67203.1 134296.1 134297.1 AD- A- usgsgcugGfgAfAfAfcaucaaaggaL962223 A- usCfscuuUfgauguuuCfcCfagccasgsg 2401 67204.1 134298.1 134299.1AD- A- gsgscuggGfaAfAfCfaucaaaggaaL96 2224 A-usUfsccuUfugauguuUfcCfcagccsasg 2402 67205.1 134300.1 134301.1 AD- A-uscsaaagGfaAfUfUfcucggaaagaL96 2225 A- usCfsuuuCfcgagaauUfcCfuuugasusg2403 67206.1 134302.1 134303.1 AD- A- usgsggaaAfcAfUfCfaaaggaauuaL962226 A- usAfsauuCfcuuugauGfuUfucccasgsc 2404 67207.1 134304.1 134305.1AD- A- gsgsaaacAfuCfAfAfaggaauucuaL96 2227 A-usAfsgaaUfuccuuugAfuGfuuuccscsa 2405 67208.1 134306.1 134307.1 AD- A-gsasaacaUfcAfAfAfggaauucucaL96 2228 A- usGfsagaAfuuccuuuGfaUfguuucscsc2406 67209.1 134308.1 134309.1 AD- A- asasacauCfaAfAfGfgaauucucgaL962229 A- usCfsgagAfauuccuuUfgAfuguuuscsc 2407 67210.1 134310.1 134311.1AD- A- csasucaaAfgGfAfAfuucucggaaaL96 2230 A-usUfsuccGfagaauucCfuUfugaugsusu 2408 67211.1 134312.1 134313.1 AD- A-asasaggaAfuUfCfUfcggaaagaaaL96 2231 A- usUfsucuUfuccgagaAfuUfccuuusgsa2409 67212.1 134314.1 134315.1 AD- A- asasggaaUfuCfUfCfggaaagaagaL962232 A- usCfsuucUfuuccgagAfaUfuccuususg 2410 67213.1 134316.1 134317.1AD- A- gsgscccuCfgAfGfAfacaagaagaaL96 2233 A-usUfscuuCfuuguucuCfgAfgggccsusu 2411 67214.1 134320.1 134321.1 AD- A-gscsccucGfaGfAfAfcaagaagaaaL96 2234 A- usUfsucuUfcuuguucUfcGfagggcscsu2412 67215.1 134322.1 134323.1 AD- A- cscscucgAfgAfAfCfaagaagaagaL962235 A- usCfsuucUfucuuguuCfuCfgagggscsc 2413 67216.1 134324.1 134325.1AD- A- gsgsaaagGfaAfAfAfgaagaguagaL96 2236 A-usCfsuacUfcuucuuuUfcCfuuuccsusc 2414 67217.1 134326.1 134327.1 AD- A-asasggaaAfaGfAfAfgaguagccgaL96 2237 A- usCfsggcUfacucuucUfuUfuccuususc2415 67218.1 134328.1 134329.1 AD- A- gsgsggguGfuGfAfAfcccccucgaaL962238 A- usUfscgaGfgggguucAfcAfcccccsasa 2416 67219.1 134330.1 134331.1AD- A- gsgsguguGfaAfCfCfcccucgaagaL96 2239 A-usCfsuucGfaggggguUfcAfcacccscsc 2417 67220.1 134332.1 134333.1 AD- A-Y44AGUUUCUUGAUCUUCUUCUUu 2240 A- AAAGAAGAAGAUCAAGAAACUUg 2418 67250.1134346.1 134347.1 AD- A- Y44UCUUUUCUGAUAAAGAGGAGa 2241 A-UCUCCUCUUUAUCAGAAAAGAGu 2419 67251.1 134348.1 134349.1 AD- A-Y44AGUGGUAAAGAGUAUUGGAAa 2242 A- UUUCCAAUACUCUUUACCACUUu 2420 67252.1134350.1 134351.1 AD- A- Y44CUCAAGUUUCUUGAUCUUCUu 2243 A-AAGAAGAUCAAGAAACUUGAGGu 2421 67253.1 134352.1 134353.1 AD- A-Y44AAGGCCAAAAGAGAAUACAAa 2244 A- UUUGUAUUCUCUUUUGGCCUUCu 2422 67254.1134354.1 134355.1 AD- A- Y44AGGCCAAAAGAGAAUACAAGa 2245 A-UCUUGUAUUCUCUUUUGGCCUUc 2423 67255.1 134358.1 134359.1 AD- A-Y44CGAUAGAGAAUCGAGAGAAAa 2246 A- UUUUCUCUCGAUUCUCUAUCGGa 2424 67256.1134360.1 134361.1 AD- A- Y44GAUAGAGAAUCGAGAGAAAAg 2247 A-CUUUUCUCUCGAUUCUCUAUCGg 2425 67257.1 134362.1 134363.1 AD- A-Y44ACCCACAAAUCUCUCUAGAUu 2248 A- AAUCUAGAGAGAUUUGUGGGUCc 2426 67258.1134364.1 134365.1 AD- A- Y44GGAUGGGAAGAGUAUAUCCUa 2249 A-UAGGAUAUACUCUUCCCAUCCGa 2427 67259.1 134366.1 134367.1 AD- A-Y44CCGAUAGAGAAUCGAGAGAAa 2250 A- UUUCUCUCGAUUCUCUAUCGGAa 2428 67260.1134368.1 134369.1 AD- A- Y44UCCGAUAGAGAAUCGAGAGAa 2251 A-UUCUCUCGAUUCUCUAUCGGAAu 2429 67261.1 134370.1 134371.1 AD- A-Y44UGAGUGGAAACCCGCUUUAUu 2252 A- AAUAAAGCGGGUUUCCACUCACa 2430 67262.1134372.1 134373.1 AD- A- Y44CCUUCUUUCCGAGAAUUCCUu 2253 A-AAGGAAUUCUCGGAAAGAAGGAc 2431 67263.1 134374.1 134375.1 AD- A-Y44CUGUGAGUGGAAACCCGCUUu 2254 A- AAAGCGGGUUUCCACUCACAGGu 2432 67264.1134376.1 134377.1 AD- A- Y44GACCCACAAAUCUCUCUAGAu 2255 A-AUCUAGAGAGAUUUGUGGGUCCc 2433 67265.1 134378.1 134379.1 AD- A-Y44GCUGGGAAACAUCAAAGGAAu 2256 A- AUUCCUUUGAUGUUUCCCAGCCa 2434 67266.1134380.1 134381.1 AD- A- Y44ACAGUUGGAAGGCUCAAGGAg 2257 A-UGUCAACCUUCCGAGUUCCUCUu 2435 67267.1 134382.1 134383.1 AD- A-Y44AAGGAGGAGGAACGAGUCCGa 2258 A- UUCCUCCUCCUUGCUCAGGCUCu 2436 67268.1134384.1 134385.1 AD- A- Y44UCUCCUCUUUAUCAGAAAAGa 2259 A-UCUUUUCUGAUAAAGAGGAGACu 2437 67269.1 134386.1 134387.1 AD- A-Y44UCCAGCAGUCUCCUCUUUAUa 2260 A- UAUAAAGAGGAGACUGCUGGACu 2438 67270.1134388.1 134389.1 AD- A- Y44AAAGAAGAAGAUCAAGAAACu 2261 A-AGUUUCUUGAUCUUCUUCUUUGu 2439 67271.1 134390.1 134391.1 AD- A-Y44AAGAAGAAGAUCAAGAAACUu 2262 A- AAGUUUCUUGAUCUUCUUCUUUg 2440 67272.1134392.1 134393.1 AD- A- Y44GAGGUCGAAAAUCCCUGGCUa 2263 A-UAGCCAGGGAUUUUCGACCUCAa 2441 67273.1 134394.1 134395.1 AD- A-Y44CCCUGGCUGGGAAACAUCAAa 2264 A- UUUGAUGUUUCCCAGCCAGGGAu 2442 67274.1134396.1 134397.1 AD- A- Y44CUGGCUGGGAAACAUCAAAGa 2265 A-UCUUUGAUGUUUCCCAGCCAGGg 2443 67275.1 134398.1 134399.1 AD- A-Y44UGGCUGGGAAACAUCAAAGGa 2266 A- UCCUUUGAUGUUUCCCAGCCAGg 2444 67276.1134400.1 134401.1 AD- A- Y44GGCUGGGAAACAUCAAAGGAa 2267 A-UUCCUUUGAUGUUUCCCAGCCAg 2445 67277.1 134402.1 134403.1 AD- A-Y44UCAAAGGAAUUCUCGGAAAGa 2268 A- UCUUUCCGAGAAUUCCUUUGAUg 2446 67278.1134404.1 134405.1 AD- A- Y44UGGGAAACAUCAAAGGAAUUa 2269 A-UAAUUCCUUUGAUGUUUCCCAGc 2447 67279.1 134406.1 134407.1 AD- A-Y44GGAAACAUCAAAGGAAUUCUa 2270 A- UAGAAUUCCUUUGAUGUUUCCCa 2448 67280.1134408.1 134409.1 AD- A- Y44GAAACAUCAAAGGAAUUCUCa 2271 A-UGAGAAUUCCUUUGAUGUUUCCc 2449 67281.1 134410.1 134411.1 AD- A-Y44CAUCAAAGGAAUUCUCGGAAa 2272 A- UUUCCGAGAAUUCCUUUGAUGUu 2450 67282.1134414.1 134415.1 AD- A- Y44AAAGGAAUUCUCGGAAAGAAa 2273 A-UUUCUUUCCGAGAAUUCCUUUGa 2451 67283.1 134416.1 134417.1 AD- A-Y44AAGGAAUUCUCGGAAAGAAGa 2274 A- UCUUCUUUCCGAGAAUUCCUUUg 2452 67284.1134418.1 134419.1 AD- A- Y44ACGAAGGAAGGCCCUCGAGAa 2275 A-UUCUCGAGGGCCUUCCUUCGUCg 2453 67285.1 134420.1 134421.1 AD- A-Y44GGCCCUCGAGAACAAGAAGAa 2276 A- UUCUUCUUGUUCUCGAGGGCCUu 2454 67286.1134422.1 134423.1 AD- A- Y44GCCCUCGAGAACAAGAAGAAa 2277 A-UUUCUUCUUGUUCUCGAGGGCCu 2455 67287.1 134424.1 134425.1 AD- A-Y44CCCUCGAGAACAAGAAGAAGa 2278 A- UCUUCUUCUUGUUCUCGAGGGCc 2456 67288.1134426.1 134427.1 AD- A- Y44GGAAAGGAAAAGAAGAGUAGa 2279 A-UCUACUCUUCUUUUCCUUUCCUc 2457 67289.1 134428.1 134429.1 AD- A-Y44AAGGAAAAGAAGAGUAGCCGa 2280 A- UCGGCUACUCUUCUUUUCCUUUc 2458 67290.1134430.1 134431.1 AD- A- Y44GGGGGUGUGAACCCCCUCGAa 2281 A-UUCGAGGGGGUUCACACCCCCAa 2459 67291.1 134432.1 134433.1 AD- A-Y44GGGUGUGAACCCCCUCGAAGa 2282 A- UCUUCGAGGGGGUUCACACCCCc 2460 67292.1134434.1 134435.1 AD- A- asasacUfuGfAfGfgucgaaaauaL96 2283 A-usAfsuuuUfcgaccucAfaGfuuususu 2461 70224.1 141079.1 141080.1 AD- A-asasacuuGfAfGfgucgaaaauaL96 2284 A- usAfsuuuucgaccucAfaGfuuususu 246270225.1 141081.1 141082.1 AD- A- asasacuuGfaGfgucgaaaauaL96 2285 A-usAfsuuuucgaccdTcAfaguuususu 2463 70226.1 141083.1 141084.1 AD- A-asasacuuGfaGfgucga(Agn)aauaL96 2286 A- usAfsuuuucgaccdTcAfaguuususu 246470227.1 141085.1 141084.1 AD- A- asGfsaaacuuGfaGfguCfgAfaaausa 2287 A-usAfsuuuucgAfcCfucAfaGfuuucsu 2465 70228.1 141086.1 141087.1 AD- A-gsasucaaGfAfAfacuugagguaL96 2288 A- usAfsccucaaguuucUfuGfaucsusu 246670229.1 141090.1 141091.1 AD- A- gsasucaaGfaAfacuugagguaL96 2289 A-usAfsccucaaguudTcUfugaucsusu 2467 70230.1 141092.1 141093.1 AD- A-gsasucaaGfaAfacuug(Agn)gguaL96 2290 A- usAfsccucaaguudTcUfugaucsusu 246870231.1 141094.1 141093.1 AD- A- asAfsgaucaaGfaAfacUfuGfaggusa 2291 A-usAfsccucaaGfuUfucUfuGfaucusu 2469 70232.1 141095.1 141096.1 AD- A-uscsaaGfaAfAfCfuugaggucgaL96 2292 A- usCfsgacCfuCfAfaguuUfcUfugasusu2470 70233.1 141097.1 141098.1 AD- A- uscsaaGfaAfAfCfuugaggucgaL96 2293A- usCfsgacCfucaaguuUfcUfugasusu 2471 70234.1 141097.1 141099.1 AD- A-uscsaagaAfAfCfuugaggucgaL96 2294 A- usCfsgaccucaaguuUfcUfugasusu 247270235.1 141100.1 141101.1 AD- A- uscsaagaAfaCfuugaggucgaL96 2295 A-usCfsgaccucaagdTuUfcuugasusu 2473 70236.1 141102.1 141103.1 AD- A-uscsaagaAfaCfuugagdCucgaL96 2296 A- usCfsgaccucaagdTuUfcuugasusu 247470237.1 141104.1 141103.1 AD- A- usCfsaagaAfaCfuugaGfgUfcgsa 2297 A-usCfsgaccucAfaGfuuUfcUfugsa 2475 70238.1 141105.1 141106.1 AD- A-asasgaUfcAfAfGfaaacuugagaL96 2298 A- usCfsucaAfgUfUfucuuGfaUfcuususu2476 70239.1 141107.1 141108.1 AD- A- asasgaUfcAfAfGfaaacuugagaL96 2299A- usCfsucaAfguuucuuGfaUfcuususu 2477 70240.1 141107.1 141109.1 AD- A-asasgaucAfAfGfaaacuugagaL96 2300 A- usCfsucaaguuucuuGfaUfcuususu 247870241.1 141110.1 141111.1 AD- A- asasgaucAfaGfaaacuugagaL96 2301 A-usCfsucaaguuucdTuGfaucuususu 2479 70242.1 141112.1 141113.1 AD- A-asasgaucAfaGfaaacu(Agn)gagaL96 2302 A- usCfsucaaguuucdTuGfaucuususu 248070243.1 141114.1 141113.1 AD- A- asGfsaagaucAfaGfaaAfcUfugagsa 2303 A-usCfsucaaguUfuCfuuGfaUfcuucsu 2481 70244.1 141115.1 141116.1 AD- A-csasucAfaAfGfGfaauucucggaL96 2304 A- usCfscgaGfaauuccuUfuGfaugsusu 248270245.1 141117.1 141118.1 AD- A- csasucaaAfGfGfaauucucggaL96 2305 A-usCfscgagaauuccuUfuGfaugsusu 2483 70246.1 141119.1 141120.1 AD- A-csasucaaAfgGfaauucucggaL96 2306 A- usCfscgagaauucdCuUfugaugsusu 248470247.1 141121.1 141122.1 AD- A- csasucaaAfgGfaauuc(Tgn)cggaL96 2307 A-usCfscgagaauucdCuUfugaugsusu 2485 70248.1 141123.1 141122.1 AD- A-asAfscaucaaAfgGfaaUfuCfucggsa 2308 A- usCfscgagaaUfuCfcuUfuGfaugusu 248670249.1 141124.1 141125.1 AD- A- asuscaAfgAfAfAfcuugaggucaL96 2309 A-usGfaccUfcAfAfguuuCfuUfgaususu 2487 70250.1 141126.1 141127.1 AD- A-asuscaAfgAfAfAfcuugaggucaL96 2310 A- usGfaccUfcaaguuuCfuUfgaususu 248870251.1 141126.1 141128.1 AD- A- asuscaagAfAfAfcuugaggucaL96 2311 A-usGfaccucaaguuuCfuUfgaususu 2489 70252.1 141129.1 141130.1 AD- A-asuscaagAfaAfcuugaggucaL96 2312 A- usGfaccucaagudTuCfuugaususu 249070253.1 141131.1 141132.1 AD- A- asuscaagAfaAfcuugadCgucaL96 2313 A-usGfaccucaagudTuCfuugaususu 2491 70254.1 141133.1 141132.1 AD- A-asGfsaucaagAfaAfcuUfgAfggucsa 2314 A- usGfaccucaAfgUfuuCfuUfgaucsu 249270255.1 141134.1 141135.1 AD- A- gsasagAfuCfAfAfgaaacuugaaL96 2315 A-usUfscaaGfuuucuugAfuCfuucsusu 2493 70256.1 141136.1 141137.1 AD- A-gsasagauCfAfAfgaaacuugaaL96 2316 A- usUfscaaguuucuugAfuCfuucsusu 249470257.1 141138.1 141139.1 AD- A- gsasagauCfaAfgaaacuugaaL96 2317 A-usUfscaaguuucudTgAfucuucsusu 2495 70258.1 141140.1 141141.1 AD- A-gsasagauCfaAfgaaac(Tgn)ugaaL96 2318 A- usUfscaaguuucudTgAfucuucsusu 249670259.1 141142.1 141141.1 AD- A- asAfsgaagauCfaAfgaAfaCfuugasa 2319 A-usUfscaaguuUfcUfugAfuCfuucusu 2497 70260.1 141143.1 141144.1 AD- A-csasaagGfaAfUfUfcucggaaagL96 2320 A- csUfuucCfgAfGfaauuCfcUfuugsasu 249870261.1 141145.1 141146.1 AD- A- csasaagGfaAfUfUfcucggaaagL96 2321 A-csUfuucCfgagaauuCfcUfuugsasu 2499 70262.1 141145.1 141147.1 AD- A-csasaaggaAfUfUfcucggaaagL96 2322 A- csUfuuccgagaauuCfcUfuugsasu 250070263.1 141148.1 141149.1 AD- A- csasaaggaAfuUfcucggaaagL96 2323 A-csUfuuccgagaadTuCfcuuugsasu 2501 70264.1 141150.1 141151.1 AD- A-csasaaggaAfuUfcucgdCaaagL96 2324 A- csUfuuccgagaadTuCfcuuugsasu 250270265.1 141152.1 141151.1 AD- A- asUfscaaaggaAfuUfcuCfgGfaaasg 2325 A-csUfuuccgaGfaAfuuCfcUfuugasu 2503 70266.1 141153.1 141154.1 AD- A-csasagAfaAfCfUfugaggucgaaL96 2326 A- usUfscgaCfcUfCfaaguUfuCfuugsasu2504 70267.1 141155.1 141156.1 AD- A- csasagAfaAfCfUfugaggucgaaL96 2327A- usUfscgaCfcucaaguUfuCfuugsasu 2505 70268.1 141155.1 141157.1 AD- A-csasagaaAfCfUfugaggucgaaL96 2328 A- usUfscgaccucaaguUfuCfuugsasu 250670269.1 141158.1 141159.1 AD- A- csasagaaAfcUfugaggucgaaL96 2329 A-usUfscgaccucaadGuUfucuugsasu 2507 70270.1 141160.1 141161.1 AD- A-csasagaaAfcUfugagg(Tgn)cgaaL96 2330 A- usUfscgaccucaadGuUfucuugsasu 250870271.1 141162.1 141161.1 AD- A- asUfscaagaaAfcUfugAfgGfucgasa 2331 A-usUfscgaccuCfaAfguUfuCfuugasu 2509 70272.1 141163.1 141164.1 AD- A-gsasagAfaGfAfUfcaagaaacuuL96 2332 A- asAfsguuUfcUfUfgaucUfuCfuucsusu2510 70273.1 141165.1 141166.1 AD- A- gsasagAfaGfAfUfcaagaaacuuL96 2333A- asAfsguuUfcuugaucUfuCfuucsusu 2511 70274.1 141165.1 141167.1 AD- A-gsasagaaGfAfUfcaagaaacuuL96 2334 A- asAfsguuucuugaucUfuCfuucsusu 251270275.1 141168.1 141169.1 AD- A- gsasagaaGfaUfcaagaaacuuL96 2335 A-asAfsguuucuugadTcUfucuucsusu 2513 70276.1 141170.1 141171.1 AD- A-gsasagaaGfaUfcaaga(Agn)acuuL96 2336 A- asAfsguuucuugadTcUfucuucsusu 251470277.1 141172.1 141171.1 AD- A- asAfsgaagaaGfaUfcaAfgAfaacusu 2337 A-asAfsguuucuUfgAfucUfuCfuucusu 2515 70278.1 141173.1 141174.1 AD- A-gsgsaaAfcAfUfCfaaaggaauuaL96 2338 A- usAfsauuCfcUfUfugauGfuUfuccsusu2516 70279.1 141175.1 141176.1 AD- A- gsgsaaAfcAfUfCfaaaggaauuaL96 2339A- usAfsauuCfcuuugauGfuUfuccsusu 2517 70280.1 141175.1 141177.1 AD- A-gsgsaaacAfUfCfaaaggaauuaL96 2340 A- usAfsauuccuuugauGfuUfuccsusu 251870281.1 141178.1 141179.1 AD- A- gsgsaaacAfuCfaaaggaauuaL96 2341 A-usAfsauuccuuugdAuGfuuucsusu 2519 70282.1 141180.1 141181.1 AD- A-gsgsaaacAfuCfaaagg(Agn)auuaL96 2342 A- usAfsauuccuuugdAuGfuuuccsusu 252070283.1 141182.1 141183.1 AD- A- usGfsggaaacAfuCfaaAfgGfaauusa 2343 A-usAfsauuccuUfuGfauGfuUfucccsa 2521 70284.1 141184.1 141185.1 AD- A-asasagaaGfaAfGfAfucaagaaacuL96 2344 A- asGfsuuuCfuUfGfaucuUfcUfucuuusgsu2522 70285.1 134288.1 141186.1 AD- A- asasagaagaAfGfAfucaagaaacuL96 2345A- asGfsuuucuugaucuUfcUfucuuusgsu 2523 70286.1 141187.1 141188.1 AD- A-asasagaagaAfgAfucaagaaacuL96 2346 A- asGfsuuucuugaudCuUfcuucuuusgsu 252470287.1 141189.1 141190.1 AD- A- asasagaagaAfgAfucaag(Agn)aacuL96 2347A- asGfsuuucuugaudCuUfcuucuuusgsu 2525 70288.1 141191.1 141190.1 AD- A-asAfsagaagaAfgAfucAfaGfaaacsu 2348 A- asGfsuuucuuGfaUfcuUfcUfucuusu 252670289.1 141192.1 141193.1 AD- A- asgsaaGfaAfGfAfucaagaaacuL96 2349 A-asGfsuuuCfuUfGfaucuUfcUfucususu 2527 70290.1 141194.1 141195.1 AD- A-asgsaaGfaAfGfAfucaagaaacuL96 2350 A- asGfsuuuCfuugaucuUfcUfucususu 252870291.1 141194.1 141196.1 AD- A- asgsaagaAfGfAfucaagaaacuL96 2351 A-asGfsuuucuugaucuUfcUfucususu 2529 70292.1 141197.1 141198.1 AD- A-asgsaagaAfgAfucaagaaacuL96 2352 A- asGfsuuucuugaudCuUfcuucususu 253070293.1 141199.1 141200.1 AD- A- asgsaagaAfgAfucaag(Agn)aacuL96 2353 A-asGfsuuucuugaudCuUfcuucususu 2531 70294.1 141201.1 141200.1 AD- A-asGfsaagaAfgAfucaaGfaAfacsu 2354 A- asGfsuuucuuGfaUfcuUfcUfucsu 253270295.1 141202.1 141203.1 AD- A- asascaUfcAfAfAfggaauucucaL96 2355 A-usGfsagaAfuUfCfcuuuGfaUfguususu 2533 70296.1 141204.1 141205.1 AD- A-asascaUfcAfAfAfggaauucucaL96 2356 A- usGfsagaAfuuccuuuGfaUfguususu 253470297.1 141204.1 141206.1 AD- A- asascaucAfAfAfggaauucucaL96 2357 A-usGfsagaauuccuuuGfaUfguususu 2535 70298.1 141207.1 141208.1 AD- A-asascaucAfaAfggaauucucaL96 2358 A- usGfsagaauuccudTuGfauguususu 253670299.1 141209.1 141210.1 AD- A- asascaucAfaAfggaau(Tgn)cucaL96 2359 A-usGfsagaauuccudTuGfauguususu 2537 70300.1 141211.1 141210.1 AD- A-asAfscaucAfaAfggaaUfuCfucsa 2360 A- usGfsagaauuCfcUfuuGfaUfgusu 253870301.1 141212.1 141213.1 AD- A- asgsgaAfuUfCfUfcggaaagaaaL96 2361 A-usUfsucuUfuCfCfgagaAfuUfccususu 2539 70302.1 141214.1 141215.1 AD- A-asgsgaAfuUfCfUfcggaaagaaaL96 2362 A- usUfsucuUfuccgagaAfuUfccususu 254070303.1 141214.1 141216.1 AD- A- asgsgaauUfCfUfcggaaagaaaL96 2363 A-usUfsucuuuccgagaAfuUfccususu 2541 70304.1 141217.1 141218.1 AD- A-asgsgaauUfcUfcggaaagaaaL96 2364 A- usUfsucuuuccgadGaAfuuccususu 254270305.1 141219.1 141220.1 AD- A- asgsgaauUfcUfcggaa(Agn)gaaaL96 2365 A-usUfsucuuuccgadGaAfuuccususu 2543 70306.1 141221.1 141220.1 AD- A-asAfsaggaauUfcUfcgGfaAfagaasa 2366 A- usUfsucuuucCfgAfgaAfuUfccuusu 254470307.1 141222.1 141223.1 AD- A- asasagGfaAfUfUfcucggaaagaL96 2367 A-usCfsuuuCfcGfAfgaauUfcCfuuususu 2545 70308.1 141224.1 141225.1 AD- A-asasagGfaAfUfUfcucggaaagaL96 2368 A- usCfsuuuCfcgagaauUfcCfuuususu 254670309.1 141224.1 141226.1 AD- A- asasaggaAfUfUfcucggaaagaL96 2369 A-usCfsuuuccgagaauUfcCfuuususu 2547 70310.1 141227.1 141228.1 AD- A-asasaggaAfuUfcucggaaagaL96 2370 A- usCfsuuuccgagadAuUfccuuususu 254870311.1 141229.1 141230.1 AD- A- asasaggaAfuUfcucgg(Agn)aagaL96 2371 A-usCfsuuuccgagadAuUfccuuususu 2549 70312.1 141231.1 141230.1 AD- A-usCfsaaaggaAfuUfcuCfgGfaaagsa 2372 A- usCfsuuuccgAfgAfauUfcCfuuugsa 255070313.1 141232.1 141233.1

TABLE 33 HDV Single dose screen in Cos7 Cells against Antigenome andGenome constructs 10 nM 1 nM 0.1 nM 10 nM 1 nM 0.1 nM 1 nM 10 nM 0.1 nMAvg Avg Avg Avg Avg Avg 10 nM SD 0.1 nM SD 1 nM SD SD Duplex Anti AntiAnti Genome Genome Genome SD Anti Anti SD Anti Genome Genome GenomeAD-67176.1 79.3 89.6 59.9 77.2 22.9 3.4 9.10 20.6 AD-67177.1 101.5 99.139.3 83.6 39.7 14.9 10.76 28.7 AD-67178.1 99.6 97.4 90.5 86.7 12.0 20.140.51 41.6 AD-67179.1 84.8 88.6 63.2 81.3 12.1 15.2 19.52 28.2AD-67180.1 77.7 108.6 41.0 93.2 15.1 26.8 13.25 33.6 AD-67181.1 169.392.9 47.9 92.3 6.2 8.9 8.51 25.0 AD-67182.1 62.1 102.6 43.6 86.3 11.422.0 12.92 28.7 AD-67183.1 89.3 105.0 65.4 99.2 13.9 14.7 4.65 34.1AD-67184.1 49.9 102.4 96.4 102.7 10.6 16.4 25.05 20.7 AD-67185.1 87.187.7 80.9 81.0 23.9 4.5 18.19 19.7 AD-67186.1 102.4 109.4 81.7 82.6 20.212.7 12.49 4.9 AD-67187.1 72.0 93.2 87.7 95.4 7.4 11.1 13.69 23.2AD-67188.1 68.1 110.5 127.7 97.3 12.8 14.9 18.70 26.5 AD-67189.1 84.395.9 104.7 98.9 16.9 15.0 23.72 26.2 AD-67190.1 104.5 115.3 110.5 101.52.7 7.2 24.53 5.1 AD-67191.1 72.9 99.8 72.8 92.2 8.4 8.1 21.61 14.0AD-67192.1 124.2 106.4 96.4 113.5 25.6 18.2 22.27 36.9 AD-67193.1 91.3105.3 105.2 121.4 12.8 21.7 25.63 41.9 AD-67194.1 39.4 84.8 97.5 105.99.0 3.1 12.31 32.6 AD-67195.1 90.1 110.7 87.5 98.7 11.7 10.4 20.22 11.7AD-67196.1 75.5 107.9 102.1 85.5 3.6 5.5 2.71 14.3 AD-67197.1 68.8 98.680.2 96.0 5.3 13.5 15.68 23.1 AD-67198.1 49.5 101.2 106.2 111.0 2.7 12.720.42 15.9 AD-67199.1 27.1 72.4 162.7 87.3 4.9 11.4 23.09 21.3AD-67200.1 24.1 76.7 128.5 114.2 4.0 4.2 13.53 24.3 AD-67201.1 58.1 88.6100.1 86.0 9.9 17.3 26.61 24.4 AD-67202.1 30.3 92.1 107.3 110.5 4.1 13.627.71 17.9 AD-67203.1 55.3 99.6 109.7 99.8 4.0 8.0 28.27 26.2 AD-67204.171.6 101.4 111.1 107.5 7.7 12.0 49.72 29.2 AD-67205.1 54.2 95.4 89.390.2 6.4 14.7 17.80 24.2 AD-67206.1 52.4 89.9 106.9 103.8 12.6 7.0 19.4736.2 AD-67207.1 57.6 99.4 102.6 109.5 11.1 21.9 9.58 9.6 AD-67208.1 31.289.5 87.4 102.7 3.5 8.9 15.91 27.3 AD-67209.1 47.1 90.6 97.8 78.5 6.310.7 18.03 25.7 AD-67210.1 31.7 95.4 96.2 92.8 1.8 14.4 14.11 28.7AD-67211.1 25.4 79.8 110.2 93.4 6.5 9.5 23.84 27.4 AD-67212.1 32.2 93.990.7 67.7 4.9 5.8 22.15 23.4 AD-67213.1 49.2 97.7 97.4 75.5 5.2 9.617.73 25.9 AD-67214.1 56.8 92.7 97.8 99.4 9.6 10.1 20.89 21.4 AD-67215.139.0 99.2 92.1 95.4 11.3 5.5 6.57 11.4 AD-67216.1 57.1 94.3 91.2 87.512.6 11.9 18.27 25.2 AD-67217.1 63.4 101.4 90.0 96.2 2.1 10.7 15.73 25.7AD-67218.1 65.8 97.3 91.1 86.6 10.3 7.1 11.42 21.2 AD-67219.1 77.3 100.483.9 89.8 1.7 7.8 18.81 40.2 AD-67220.1 78.8 102.2 87.6 77.8 15.2 7.97.96 11.5 AD-67250.1 85.3 92.4 29.0 74.3 4.4 11.6 3.72 22.8 AD-67251.190.2 104.3 101.9 91.7 25.6 18.0 25.07 12.2 AD-67252.1 99.6 108.6 59.664.9 8.2 3.6 18.48 13.7 AD-67253.1 50.0 94.6 32.9 73.5 3.3 15.8 14.4418.2 AD-67254.1 193.2 106.8 63.7 68.2 47.1 6.1 22.95 12.7 AD-67255.183.7 94.5 38.2 66.7 25.7 13.7 8.71 9.5 AD-67256.1 88.8 94.3 113.1 106.022.8 9.4 37.17 26.3 AD-67257.1 51.8 101.2 105.6 93.7 5.2 4.6 26.52 22.3AD-67258.1 81.6 102.7 92.2 92.4 24.4 6.2 39.25 26.5 AD-67259.1 113.1104.8 81.2 71.9 18.8 16.7 30.40 9.6 AD-67260.1 52.1 112.2 67.4 74.6 8.18.8 4.21 10.8 AD-67261.1 73.8 96.2 96.1 99.5 15.5 9.1 7.96 17.8AD-67262.1 110.6 106.5 127.0 115.6 28.0 15.3 20.20 3.0 AD-67263.1 69.496.3 49.7 63.1 16.7 8.8 4.00 14.8 AD-67264.1 180.6 104.6 100.1 94.9 43.222.3 37.56 10.7 AD-67265.1 122.5 117.0 119.1 99.4 23.3 19.7 13.26 18.6AD-67266.1 24.3 59.3 115.8 83.6 1.0 3.8 34.70 14.0 AD-67267.1 83.4 113.892.6 82.7 13.9 11.8 26.19 10.6 AD-67268.1 99.2 101.2 81.0 94.9 14.9 11.121.50 22.4 AD-67269.1 42.9 81.7 179.2 86.0 6.6 6.5 50.94 13.2 AD-67270.133.9 85.7 125.2 85.6 5.9 10.8 24.45 12.7 AD-67271.1 29.6 53.4 156.0115.8 8.9 10.9 27.82 27.2 AD-67272.1 13.4 46.3 136.9 128.4 2.3 4.4 23.5230.3 AD-67273.1 15.9 57.5 105.1 82.1 4.6 4.4 26.59 13.3 AD-67274.1 25.860.4 86.8 93.4 2.0 10.2 14.89 22.4 AD-67275.1 46.5 75.4 131.1 94.3 12.39.5 46.37 18.0 AD-67276.1 20.8 59.4 87.7 88.0 2.2 4.4 10.78 18.2AD-67277.1 17.2 49.5 93.4 92.4 2.3 4.7 44.06 19.4 AD-67278.1 25.1 61.3131.5 94.8 8.4 6.8 30.75 10.0 AD-67279.1 21.9 78.3 111.7 126.9 4.8 10.819.15 15.0 AD-67280.1 32.8 78.1 154.7 109.4 4.3 9.1 52.47 29.4AD-67281.1 32.8 60.0 163.7 110.1 2.4 9.0 20.60 8.6 AD-67282.1 19.3 47.6129.1 109.7 4.8 9.1 18.16 21.2 AD-67283.1 34.5 71.8 193.2 112.4 14.0 1.166.11 10.7 AD-67284.1 51.2 76.9 126.9 94.1 5.2 7.9 40.77 26.8 AD-67285.117.3 68.6 66.9 110.7 1.9 10.7 26.05 25.3 AD-67286.1 29.5 85.5 87.3 125.75.8 10.7 13.51 30.7 AD-67287.1 33.1 83.5 84.9 98.8 12.2 22.2 33.27 12.9AD-67288.1 14.4 75.1 73.3 109.2 6.5 5.2 15.93 16.1 AD-67289.1 38.8 66.497.9 102.0 15.6 7.9 22.42 19.0 AD-67290.1 20.9 81.2 89.6 112.4 4.8 9.311.83 8.4 AD-67291.1 47.8 100.0 107.1 95.2 4.7 19.7 16.82 9.3 AD-67292.199.9 99.3 129.3 136.5 22.7 20.8 39.06 58.4 AD-70224.1 46.5 68.6 94.099.6 113.6 103.5 10.2 8.7 2.5 13.9 6.9 10.0 AD-70225.1 49.1 76.2 88.9129.9 107.0 87.1 7.2 0.8 5.3 45.3 6.1 10.0 AD-70226.1 65.3 105.4 8.023.9 AD-70227.1 37.7 58.7 84.3 173.3 111.4 101.7 8.2 3.2 5.1 15.5 6.25.5 AD-70228.1 102.4 120.8 107.9 46.3 52.2 82.3 10.7 5.7 14.3 11.4 2.53.6 AD-70229.1 32.0 65.6 96.9 92.1 107.2 89.6 3.5 0.1 1.9 6.8 25.7 5.5AD-70230.1 36.7 66.7 99.2 91.8 100.1 91.9 3.9 11.4 13.6 8.2 14.9 12.5AD-70231.1 26.7 62.6 86.3 97.3 93.9 122.5 4.0 5.2 2.4 25.6 2.7 35.5AD-70232.1 16.1 35.2 74.5 40.1 33.3 73.9 2.7 2.7 0.9 15.0 6.5 10.9AD-70233.1 24.8 71.7 96.0 91.8 106.4 96.2 1.6 1.0 1.2 10.4 9.2 0.5AD-70234.1 29.6 63.0 90.2 89.4 102.5 99.9 5.1 0.1 3.5 8.5 3.4 6.3AD-70235.1 35.4 84.5 104.2 66.6 101.9 97.8 10.5 4.2 9.7 13.0 1.2 6.2AD-70236.1 39.4 64.8 95.2 86.3 88.3 94.0 3.7 3.0 0.6 5.7 3.8 6.7AD-70237.1 22.1 49.4 83.8 96.2 90.3 87.6 2.5 0.1 1.7 19.1 9.4 2.8AD-70238.1 26.5 61.4 91.8 60.9 72.9 82.7 1.6 0.8 6.7 6.8 5.4 1.9AD-70239.1 73.7 99.1 8.4 20.7 AD-70240.1 80.0 82.4 11.0 8.3 AD-70241.182.5 107.4 2.1 21.9 AD-70242.1 69.5 90.2 4.7 28.9 AD-70243.1 33.5 57.4102.5 109.3 106.4 80.7 3.5 1.2 8.2 20.7 17.0 2.8 AD-70244.1 78.5 93.399.9 22.6 29.3 85.9 6.2 1.8 4.5 4.2 5.0 12.2 AD-70245.1 44.7 71.2 83.2109.7 91.5 98.5 3.3 2.4 1.5 15.1 3.9 8.9 AD-70246.1 42.1 79.0 104.6 97.299.0 97.0 1.0 4.9 13.5 25.8 4.7 6.9 AD-70247.1 60.3 75.2 6.5 6.8AD-70248.1 20.9 45.2 82.2 86.5 121.9 98.2 2.1 6.9 3.8 10.0 3.5 2.4AD-70249.1 72.4 93.6 96.7 39.9 38.5 81.3 4.2 6.7 1.5 13.1 7.4 10.1AD-70250.1 35.0 72.9 97.5 81.7 91.4 89.8 3.6 3.1 16.8 8.5 2.0 5.2AD-70251.1 47.2 71.8 92.6 87.6 101.5 105.3 5.5 6.5 0.8 15.2 1.3 11.8AD-70252.1 55.7 76.2 6.7 6.1 AD-70253.1 61.5 68.8 3.9 16.5 AD-70254.130.0 65.2 93.1 83.1 92.0 111.6 0.8 1.4 2.6 9.8 15.2 0.8 AD-70255.1 65.089.1 90.4 32.6 50.7 92.0 5.2 1.6 2.6 6.9 8.8 2.0 AD-70256.1 17.9 45.989.0 107.7 126.3 95.6 2.3 0.6 6.6 17.1 24.6 5.3 AD-70257.1 19.7 41.878.1 129.4 104.7 96.7 3.8 0.7 3.3 28.2 16.6 3.9 AD-70258.1 25.9 51.483.8 120.5 104.3 77.0 2.6 3.8 8.2 25.8 0.2 7.1 AD-70259.1 15.0 26.0 70.3131.5 117.4 100.5 1.7 0.2 4.0 16.7 3.8 1.0 AD-70260.1 21.9 43.2 85.444.7 39.3 103.6 4.3 1.3 6.4 9.1 0.9 6.7 AD-70261.1 83.1 82.1 4.2 10.7AD-70262.1 85.6 81.5 7.2 13.1 AD-70263.1 92.8 116.9 4.7 50.7 AD-70264.182.3 120.8 5.5 53.7 AD-70265.1 38.1 72.2 95.2 130.9 98.6 90.5 4.1 1.73.1 29.9 0.8 12.6 AD-70266.1 85.8 90.3 11.3 19.5 AD-70267.1 15.0 27.866.1 123.5 122.8 103.0 1.3 1.7 2.8 15.5 4.1 8.8 AD-70268.1 15.8 30.378.2 116.7 123.1 103.6 2.0 1.8 1.8 22.7 6.9 5.3 AD-70269.1 16.0 27.971.1 106.9 99.8 99.2 1.7 1.7 0.6 19.5 9.4 26.9 AD-70270.1 26.1 54.7 89.1108.8 102.8 93.4 4.5 4.4 10.6 31.1 1.6 14.2 AD-70271.1 15.3 26.8 62.8115.4 125.0 106.9 3.3 2.3 4.3 13.5 6.9 3.7 AD-70272.1 15.1 33.3 82.353.3 31.4 70.3 2.7 2.1 11.2 15.0 5.3 12.4 AD-70273.1 26.8 49.5 85.2150.6 133.9 111.2 4.9 6.4 2.1 36.0 0.4 0.6 AD-70274.1 23.2 36.5 87.3177.6 123.0 109.8 2.8 0.5 0.6 27.1 2.2 21.5 AD-70275.1 20.0 43.9 76.1178.4 151.1 103.7 4.1 4.7 3.0 32.2 10.4 8.1 AD-70276.1 32.1 43.6 84.1183.4 130.6 103.7 1.8 2.3 2.1 14.9 12.2 5.0 AD-70277.1 29.7 33.8 73.2213.9 140.7 109.2 6.4 4.9 2.8 20.5 8.8 9.7 AD-70278.1 25.2 41.4 73.657.8 56.0 95.3 5.9 1.1 5.7 12.1 2.6 4.7 AD-70279.1 79.8 135.6 7.4 22.9AD-70280.1 100.4 162.0 14.5 17.5 AD-70281.1 82.2 161.7 13.1 10.6AD-70282.1 105.1 116.2 5.3 16.9 AD-70283.1 64.4 134.4 7.7 23.3AD-70284.1 95.4 106.1 11.0 5.3 AD-70285.1 91.6 119.4 11.6 7.9 AD-70286.184.0 110.3 8.8 21.6 AD-70287.1 89.9 96.7 4.4 7.5 AD-70288.1 29.1 74.089.9 199.1 132.5 110.7 5.8 7.8 2.7 58.5 26.6 2.8 AD-70289.1 32.7 44.484.5 77.7 60.0 76.8 5.3 1.4 2.6 11.4 5.7 1.4 AD-70290.1 102.0 102.6 13.617.4 AD-70291.1 100.7 110.1 8.5 24.6 AD-70292.1 76.7 131.9 5.2 20.2AD-70293.1 99.7 131.7 5.8 29.7 AD-70294.1 103.1 124.4 5.4 35.4AD-70295.1 101.8 125.9 6.3 10.4 AD-70296.1 104.3 106.8 11.1 11.9AD-70297.1 105.2 105.8 6.0 38.9 AD-70298.1 103.1 99.9 5.0 19.1AD-70299.1 108.1 100.9 12.9 24.6 AD-70300.1 109.6 80.5 13.3 8.3AD-70301.1 99.4 97.2 7.0 11.0 AD-70302.1 101.9 104.6 17.2 27.3AD-70303.1 77.1 133.0 6.4 27.0 AD-70304.1 119.5 108.7 12.4 10.3AD-70305.1 91.1 115.5 8.2 22.6 AD-70306.1 103.7 140.3 15.5 19.8AD-70307.1 75.4 98.0 3.8 14.0 AD-70308.1 105.5 136.1 7.7 30.9 AD-70309.1109.5 92.8 23.5 14.6 AD-70310.1 111.6 109.7 11.5 27.1 AD-70311.1 101.8111.5 10.2 25.1 AD-70312.1 113.8 91.0 8.3 26.1 AD-70313.1 114.8 76.910.0 13.1

TABLE 34 Dose response screen in Cos7 cells Antigenome GenomeAntigenome, Genome, hot spot, hot spot, Duplex IC50 nM IC50 nM IC50 nMIC50 nM AD-67176.1 not achieved not achieved AD-67177.1 not achieved notachieved AD-67180.1 not achieved 3124.5 AD-67181.1 not achieved >10000AD-67182.1 not achieved >10000 AD-67199.1 288.0 not achieved AD-67200.1466.2 not achieved AD-67202.1 >10000 not achieved AD-67208.1 notachieved not achieved AD-67210.1 >10000 not achieved AD-67211.1 1100.8not achieved AD-70228.1 not achieved 23.0 AD-70232.1 405.4 5002.4 33.79.0 AD-70238.1 273.9 122.4 AD-70244.1 1589.9 not achieved 14.7AD-70249.1 2377.2 2376.2 13.1 AD-70255.1 3082.4 26.1 AD-70259.1 173.2AD-70260.1 419.2 7185.0 50.3 8.7 AD-70266.1 not achieved 476.4AD-70267.1 185.7 AD-70268.1 174.6 AD-70269.1 267.6 AD-70271.1 119.6AD-70272.1 364.8 3400.4 30.2 22.2 AD-70274.1 258.8 AD-70277.1 190.6AD-70278.1 57.1 30.6 AD-70284.1 not achieved 4670.5 AD-70289.1 notachieved not achieved AD-70295.1 61.8 39.4 AD-70301.1 not achieved notachieved AD-70307.1 2329.6 379.0 AD-70313.1 not achieved not achieved

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments and methods described herein. Such equivalents are intendedto be encompassed by the scope of the following claims.

1. A double stranded RNAi agent for inhibiting expression of hepatitis Dvirus (HDV) in a cell, wherein said double stranded RNAi agent comprisesa sense strand and an antisense strand forming a double-stranded regionselected from the group consisting of (a) a sense strand comprises atleast 15 contiguous nucleotides differing by no more than 3 nucleotidesfrom the nucleotide sequence of SEQ ID NO:29, and said antisense strandcomprises at least 15 contiguous nucleotides differing by no more than 3nucleotides from the nucleotide sequence of SEQ ID NO:30; (b) a sensestrand comprises at least 15 contiguous nucleotides differing by no morethan 3 nucleotides from the nucleotide sequence of SEQ ID NO:31, andsaid antisense strand comprises at least 15 contiguous nucleotidesdiffering by no more than 3 nucleotides from the nucleotide sequence ofSEQ ID NO:32; (c) a sense strand comprises at least 15 contiguousnucleotides differing by no more than 3 nucleotides from the nucleotidesequence of SEQ ID NO:33, and said antisense strand comprises at least15 contiguous nucleotides differing by no more than 3 nucleotides fromthe nucleotide sequence of SEQ ID NO:34; (d) a sense strand comprises atleast 15 contiguous nucleotides differing by no more than 3 nucleotidesfrom the nucleotide sequence of SEQ ID NO:35, and said antisense strandcomprises at least 15 contiguous nucleotides differing by no more than 3nucleotides from the nucleotide sequence of SEQ ID NO:36; (e) a sensestrand comprises at least 15 contiguous nucleotides differing by no morethan 3 nucleotides from the nucleotide sequence of SEQ ID NO:37, andsaid antisense strand comprises at least 15 contiguous nucleotidesdiffering by no more than 3 nucleotides from the nucleotide sequence ofSEQ ID NO:38; (f) a sense strand comprises at least 15 contiguousnucleotides differing by no more than 3 nucleotides from the nucleotidesequence of SEQ ID NO:39, and said antisense strand comprises at least15 contiguous nucleotides differing by no more than 3 nucleotides fromthe nucleotide sequence of SEQ ID NO:40; (g) a sense strand comprises atleast 15 contiguous nucleotides differing by no more than 3 nucleotidesfrom the nucleotide sequence of SEQ ID NO:41, and said antisense strandcomprises at least 15 contiguous nucleotides differing by no more than 3nucleotides from the nucleotide sequence of SEQ ID NO:42; (h) a sensestrand comprises at least 15 contiguous nucleotides differing by no morethan 3 nucleotides from the nucleotide sequence of SEQ ID NO:43, andsaid antisense strand comprises at least 15 contiguous nucleotidesdiffering by no more than 3 nucleotides from the nucleotide sequence ofSEQ ID NO:44; and (i) a sense strand comprises at least 15 contiguousnucleotides differing by no more than 3 nucleotides from the nucleotidesequence of SEQ ID NO:2551, and said antisense strand comprises at least15 contiguous nucleotides differing by no more than 3 nucleotides fromthe nucleotide sequence of SEQ ID NO:2552; wherein substantially all ofthe nucleotides of said sense strand and substantially all of thenucleotides of said antisense strand are modified nucleotides, whereinsaid sense strand is conjugated to a ligand attached at the 3′-terminus,and wherein the ligand is one or more GalNAc derivatives attachedthrough a bivalent or trivalent branched linker.
 2. (canceled) 3.(canceled)
 4. The double stranded RNAi agent of claim 1, wherein all ofthe nucleotides of said sense strand and all of the nucleotides of saidantisense strand are modified nucleotides.
 5. (canceled)
 6. The doublestranded RNAi agent of claim 1, wherein said sense strand and saidantisense strand comprise a region of complementarity which comprises atleast 15 contiguous nucleotides differing by no more than 3 nucleotidesfrom any one of the sense and antisense strands of AD-70260.1,AD-70232.1, AD-70249.1, AD-70244.1, AD-70272.1, AD-70228.1, AD-70255.1,AD-70278.1, AD-70295.1, AD-67200.1, AD-67211.1, AD-67199.1, AD-67202.1,AD-67208.1, AD-67210.1, AD-70259.1, AD-70267.1, AD-70272.1, AD-70271.1,AD-70268.1, AD-70269.1, AD-70232.1, AD-70256.1, AD-70257.1, andAD-70275.1.
 7. (canceled)
 8. The double stranded RNAi agent of claim 1,wherein at least one of said modified nucleotides is selected from thegroup consisting of a 3′-terminal deoxy-thymine (dT) nucleotide, a2′-O-methyl modified nucleotide, a 2′-fluoro modified nucleotide, a2′-deoxy-modified nucleotide, a locked nucleotide, an unlockednucleotide, a conformationally restricted nucleotide, a constrainedethyl nucleotide, an abasic nucleotide, a 2′-amino-modified nucleotide,a 2′-O-allyl-modified nucleotide, 2′-C-alkyl-modified nucleotide,2′-hydroxyl-modified nucleotide, a 2′-methoxyethyl modified nucleotide,a 2′-O-alkyl-modified nucleotide, a morpholino nucleotide, aphosphoramidate, a non-natural base comprising nucleotide, atetrahydropyran modified nucleotide, a 1,5-anhydrohexitol modifiednucleotide, a cyclohexenyl modified nucleotide, a nucleotide comprisinga phosphorothioate group, a nucleotide comprising a methylphosphonategroup, a nucleotide comprising a 5′-phosphate, and a nucleotidecomprising a 5′-phosphate mimic.
 9. The double stranded RNAi agent ofany claim 1, wherein at least one strand comprises a 3′ overhang of atleast 1 nucleotide; or at least 2 nucleotides.
 10. (canceled)
 11. Thedouble stranded RNAi agent of claim 1, wherein the double-strandedregion is 15-30 nucleotide pairs in length; 17-23 nucleotide pairs inlength; 17-25 nucleotide pairs in length; 23-27 nucleotide pairs inlength; 19-21 nucleotide pairs in length; or 21-23 nucleotide pairs inlength. 12.-16. (canceled)
 17. The double stranded RNAi agent of claim1, wherein each strand has 15-30 nucleotides; or 19-30 nucleotides. 18.(canceled)
 19. The double stranded RNAi agent of claim 1, wherein theligand is


20. The double stranded RNAi agent of claim 1, wherein the RNAi agent isconjugated to the ligand as shown in the following schematic

wherein X is O or S.
 21. (canceled)
 22. The double stranded RNAi agentof claim 1, wherein said RNAi agent is selected from the consisting ofany one of AD-70260.1, AD-70232.1, AD-70249.1, AD-70244.1, AD-70272.1,AD-70228.1, AD-70255.1, AD-70278.1, AD-70295.1, AD-67200.1, AD-67211.1,AD-67199.1, AD-67202.1, AD-67208.1, AD-67210.1, AD-70259.1, AD-70267.1,AD-70272.1, AD-70271.1, AD-70268.1, AD-70269.1, AD-70232.1, AD-70256.1,AD-70257.1, or AD-70275.1.
 23. A double stranded RNAi agent forinhibiting expression of hepatitis D virus (HDV) in a cell, wherein saiddouble stranded RNAi agent comprises a sense strand and an antisensestrand forming a double-stranded region, wherein said sense strandcomprises any one of the sense sequences provided in any one of Table11, 12, 31, and 32, and said antisense strand comprises any one of theantisense sequences provided in any one of Table 11, 12, 31, and 32,wherein substantially all of the nucleotides of said sense strand andsubstantially all of the nucleotides of said antisense strand aremodified nucleotides, wherein said sense strand is conjugated to aligand attached at the 3′-terminus, and wherein the ligand is one ormore GalNAc derivatives attached through a bivalent or trivalentbranched linker. 24.-38. (canceled)
 39. A composition for inhibitingexpression of hepatitis D virus (HDV) in a cell, said compositioncomprising: (a) a first double-stranded RNAi agent comprising a firstsense strand and a first antisense strand forming a double-strandedregion, wherein substantially all of the nucleotides of said first sensestrand and substantially all of the nucleotides of said first antisensestrand are modified nucleotides, wherein said first sense strand isconjugated to a ligand attached at the 3′-terminus, and wherein theligand is one or more GalNAc derivatives attached through a bivalent ortrivalent branched linker; and (b) a second double-stranded RNAi agentcomprising a second sense strand and a second antisense strand forming adouble-stranded region, wherein substantially all of the nucleotides ofsaid second sense strand and substantially all of the nucleotides ofsaid second antisense strand are modified nucleotides, wherein saidsecond sense strand is conjugated to a ligand attached at the3′-terminus, and wherein the ligand is one or more GalNAc derivativesattached through a bivalent or trivalent branched linker; wherein thefirst and second sense strands each independently comprise a sequenceselected from the group consisting of any one of the sense sequences ofAD-70260.1, AD-70232.1, AD-70249.1, AD-70244.1, AD-70272.1, AD-70228.1,AD-70255.1, AD-70278.1, AD-70295.1, AD-67200.1, AD-67211.1, AD-67199.1,AD-67202.1, AD-67208.1, AD-67210.1, AD-70259.1, AD-70267.1, AD-70272.1,AD-70271.1, AD-70268.1, AD-70269.1, AD-70232.1, AD-70256.1, AD-70257.1,or AD-70275.1, and wherein the first and second antisense strands eachindependently comprise a sequence selected from the group consisting ofany one of the antisense sequences of AD-70260.1, AD-70232.1,AD-70249.1, AD-70244.1, AD-70272.1, AD-70228.1, AD-70255.1, AD-70278.1,AD-70295.1, AD-67200.1, AD-67211.1, AD-67199.1, AD-67202.1, AD-67208.1,AD-67210.1, AD-70259.1, AD-70267.1, AD-70272.1, AD-70271.1, AD-70268.1,AD-70269.1, AD-70232.1, AD-70256.1, AD-70257.1, or AD-70275.1. 40.-43.(canceled)
 44. A double stranded RNAi agent comprising the sense strandnucleotide sequence and the antisense strand nucleotide sequence of anyone of the RNAi agents provided in any one of Tables 11, 12, 31, and 32.45. (canceled)
 46. (canceled)
 47. A pharmaceutical compositioncomprising the double stranded RNAi agent of claim 1 or 23, or thecomposition of claim 31 or
 39. 48.-52. (canceled)
 53. A method ofinhibiting Hepatitis D virus (HDV) gene expression in a cell, the methodcomprising: (a) contacting the cell with the double stranded RNAi agentof claim 1 or 23, or the composition of claim 39, or the pharmaceuticalcomposition of claim 47; and (b) maintaining the cell produced in step(a) for a time sufficient to obtain degradation of the mRNA transcriptof an HDV gene, thereby inhibiting expression of the HDV gene in thecell. 54.-69. (canceled)
 70. A method of treating a subject having aHepatitis D virus (HDV) infection, comprising administering to thesubject a therapeutically effective amount of the double stranded RNAiagent of claim 1 or 23, or the composition of claim 39, or thepharmaceutical composition of claim 47, thereby treating said subject.71.-103. (canceled)
 104. The method of claim 70, further comprisingadministering to the subject an additional therapeutic agent. 105.-111.(canceled)